3. Materials and Methods
3.1. Culture and maintenance of Earthworms:
Eudrilus eugeniae and Perionyx excavatus were collected from the nearby earthworm
farm and they were maintained in separate plastic tubs containing soil, cow dung and
decayed leaf litters in the ratio of 1:4:2. The earthworms were kept in the laboratory made
condition for a minimum of ten days in order to allow them to adapt to experimental
conditions (28 ± 2°C) and to get acclimatization of the laboratory condition.
3.2. Dissection of earthworm, E.eugeniae:
The earthworm, E.eugeniae was fixed with 10% formaldehyde solution for 15
minutes on a petri plate. After fixation, the worm was taken out from the petri plate and kept
on a dissection board with dorsal up position. Using the sterile surgical blade (Blade No: 11)
the earthworm was symmetrically dissected out. Each organ from the dissected worm was
documented using digital camera.
3.3. Surgical procedure for the earthworm, E.eugeniae:
For the surgical purpose earthworms of E.eugeniae was taken in a separate beaker
and washed with the running tap water to remove any debris that are adhering to it. The
matured earthworm of well defined clitellum and prostate gland of similar sizes ranging from
12cm to 18cm were chosen because the young matured worm is having a higher growth rate
and it is well suited for the regeneration studies. For the surgical purpose the earthworm was
first kept in ice cold 1X PBS to inactivate their movement temporally and they were
positioned on the wooden plate dorsally to expose for microsurgery. The earthworm was
amputated in two different patterns. In the first case the earthworm was amputated in preclitellar portion, and in the second case the earthworm was amputated within the clitellar
portion. After microsurgery the amputated animals were allowed to recover and were kept
alive for more than three months and were monitored frequently.
39
In order to understand the regeneration process in the context of presence of intact
clitellum, partial clitellum and in the absence of the clitellum, the earthworms were grouped
into four sets and each set have three worms. In all the groups, the worms were initially
amputated at the tail segments (90th segment) and were kept for regeneration until the 5th day.
After the 5th day of post-amputation one group of worm was amputated at pre-clitellum
segment, the second group was amputated within the clitellum, the third group was
amputated at post-clitellar segments and the last group remains undisturbed and which act as
a control. The four groups of different sites amputated worm were carefully monitored on
day 5th, 9th and 13th day for the size of the anterior and posterior bud.
3.4. Surgical procedures for the earthworm, P.excavatus:
The P.excavatus with well defined clitellum and in similar length (12-14cm) was
chosen for amputation. The collected earthworm was initially washed with tap water and they
were kept in ice cold 1X PBS to inactivate their movement temporally and they were
positioned on the wooden plate dorsally to expose for microsurgery. Then the earthworm was
carefully amputated in four different patterns. The first group of worm was amputated at preclitellum segment (10th segment). Second and third group of worm was amputated at
clitellum segments (15th segment) and in post clitellum segments (25th segment) respectively.
The fourth set of worm was amputated at multiple sites at post clitellum segments. The
amputated earthworm are monitored frequently for the regeneration of the regenerative bud.
In order to understand the stored riboflavin in the coelomic fluid of P.excavatus worm
and to understand their effective utilization at the time of regeneration the worm are
simultaneously amputated at 10th and 90th segment and after the 5th day of regeneration they
were amputated again before forming the mouth and anus. The experiment was repeated
likewise for 7 times with regular examining their coelomic fluid for riboflavin presence..
40
3.5. Injection of thymidine to the earthworm, E.eugeniae and P.excavatus:
The mechanism of Morphalaxis and Epimorphosis are easily understood by injecting
2mM thymidine (an S phase blocking agent) to the living system at the time of regeneration.
For this purpose, healthy worm of E.eugeniae (1.00 g to 1.2 g) and P.excavatus (0.6 g to 0.8
g) with similar sizes were carefully selected for experiments. From the 2mM thymidine stock
solution both the earthworms, E.eugeniae and P.excavatus were injected with 10µl and 6µl
for every day before and after the amputation, respectively. The injection of 2mM thymidine
starts exactly two days before the amputation because thymidine has its effect only after
14hrs and injection was continued up to 8 days after post-amputation. The thymidine solution
was injected to the earthworm at the ventral side of 20th to the 24th segment of the worm with
30 gauge needle. Similar types of injection were performed with sterile water for control
purpose.
3.6. Surgical procedure for prostate gland amputation in the earthworm, E.eugeniae:
For the surgical purpose of prostate gland the young matured earthworms of E.
eugeniae were chosen because the growth rate of this animal is greater than well matured old
worms. For the surgical purpose the earthworm was first kept in ice cold 1X PBS to
inactivate their movement temporally and they were positioned on the wooden plate ventrally
to expose their prostate gland during microsurgery. Earthworm, E.eugeniae have one pair of
tubular prostrate which start from 19th segment and ends in 24th segment. The prostate gland
was amputated from the earthworm in three different patterns. In the first and second case
whole part of one prostate and half part of one prostate only amputated with keeping intact of
another prostate and intestinal portion to increase the survival rate and to speed up the
process of prostate regeneration. In the third case the earthworm was amputated vertically at
the 19th segment to remove the two prostate gland at a time with complete removal of
41
intestine region. After microsurgery the amputated animals were allowed to recover and were
kept alive for more than three months and were monitored frequently.
3.7. Histology:
Principle:
Histology is the technique used to study complete architecture of the tissue patterns
by means of examination and analysis of cell/tissue physiology & morphology at the
microscopic level. The principle behind the histological technique is to the preserve
microscopic anatomy of tissue and make them hard, so that very thin section (5 to 7 microns)
can be made. After staining, the section should represent the anatomy of the tissue as close to
as possible to their structure in life. This is achieved by passing the total or selected part of
the tissue through a series of process described below.
Materials:
Earthworm tissues - Normal Head Segments, 2nd, 4th, 8th, 10th, 12th, 24th day
regenerated Head segments which are amputated at 10th, 15th segment, normal clitellum
segments and clitellum segments of 3rd day Regenerating worm of E.eugeniae. Similar sets
of regenerating and normal tissues samples of P.excavatus worm are also carried out.
Microtome
Screw cap bottles
Clean Albumenized glass slides
Coupling jars
Cover slips
Forceps
Reagents used for Histology:
Fixation:
10% Formaldehyde solution was used as a fixative
42
Dehydration:
Different grades of isopropyl alcohol were used for dehydrating the tissues as follows:
60% isopropyl alcohol
70% isopropyl alcohol
80% isopropyl alcohol
90% isopropyl alcohol
100% isopropyl alcohol
100% isopropyl alcohol (fresh)
Clearing:
Xylene
Impregnation with wax:
Paraffin wax – 1
Paraffin wax – 2
Paraffin wax – 3
Reagents used for processing earthworm tissue sections:
Dewaxing:
Xylene
Rehydration:
100% isopropyl alcohol
Staining agents:
Basic dye Haematoxylin
Acidic dye Eosin
Acid alcohol
Acid alcohol was prepared by mixing 99 ml of 70% isopropyl alcohol with 1 ml of
concentrated Hydrochloric acid.
43
Decolourizer:
100% isopropyl alcohol
Methodology:
Fixation and processing of earthworm Tissue for Paraffin Sections needs the following steps
A. Fixation of Tissues in 10% Formaldehyde Solution:
1. The earthworm was cleanly washed with tap water to remove any dirty on its body.
2. Sacrifice the earthworm in 10% Formaldehyde Solution for 5 minutes. After a sacrifice
cut the desire tissues into pieces not larger than 3-5cm length.
3. The tissues were then put in 50% Formaldehyde Solution in 60º C for 24 hours for
fixation.
B. Processing:
1. After fixation, the tissue was washed with distilled water and put in 60% Iso-propyl
alcohol for 1 hour for dehydration. Similarly the steps were performed in 70%, 80%, 90%
and 100 % (two times) each at incubation of 1 hour at 60º C temperature.
2. After dehydration, the tissues were allowed for clearing the ethanol by put in xylene for 45
min.
C. Embedding of tissues in Paraffin wax:
1. After clearing, the tissues were put in a wax 1 at 60º C for 2 hours then transfer the tissues
to wax 2 for overnight and in wax 3 each for 2 hours.
2. Using two L-shaped equipment a paraffin block was prepared and in that the processes
tissue was embedded for sectioning.
44
Table 1: Steps followed in histology
Step
Temperature
Time
Fixation - 10% formaldehyde
50οC
24 hours
Dehydration- 60% isopropyl alcohol
50οC
1 hour
Dehydration- 70% isopropyl alcohol
50οC
1 hour
Dehydration- 80% isopropyl alcohol
50οC
1 hour
Dehydration- 90% isopropyl alcohol
50οC
Overnight
Dehydration- 100% isopropyl alcohol
50οC
1 hour
Dehydration- 100% isopropyl alcohol
50οC
1 hour
Clearing – Xylene
50οC
1 hour
Wax impregnation – Paraffin Wax 1
58-60οC
Overnight
Wax impregnation – Paraffin Wax 2
58-60οC
3 hours
Wax impregnation – Paraffin Wax 3
58-60οC
3 hours
D. Sectioning:
1. Section paraffin blocks at desires thickness (usually 5-7µm) on a microtome and the
ribbon was carefully transferred to an Egg albumin: Glycerol (50:50) coated slide overlaid
with distilled water.
2. Place the slide in 60º C for half an hour to melt the paraffin then deparaffinize slides
in
two changes of xylene for 5 minutes each.
45
3. Transfer the slide to isopropyl alcohol for 2 minutes before washing the deparaffinize
slides in water.
E. Staining:
1. The deparaffinized slide washed in water was stained with haematoxylin (which stains
nucleus) for 6-8 minutes and the excess stains were removed by water wash followed by acid
alcohol treatment (99 ml of 70% isopropyl alcohol and 1ml of concentrated HCL) for 30
seconds.
2. The excess stains were further removed by keeping the slide for blooming in running tap
water for overnight.
3. The nucleus stained slide was counter stained with eosin (which stains cytoplasm) for 30
seconds and the excess stains were removed by dipping in water and isopropyl alcohol.
4. Then the slide was dipped in xylene and the slides were mounted by overlaid with DPX
followed by careful plotting of the cover slip. The setup is undisturbed for 1 days to dry and
after that it is ready for microscopic examination.
3.8. Lowry’s method of protein estimation:
Principle:
Protein can be estimated by different methods, the most popular method was
described by Lowry et al., (1951). First the proteins are pre-treated with copper ion in alkali
solution. Under alkaline conditions cupric ions (Cu2+) chelate with the peptide bonds
resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu+). The Cuprous ions can also
be detected with Folin Ciocalteu Reagent. The aromatic amino acids (tyrosine and
tryptophan) in the treated sample, reduce the phosphomolybdatephosphotungustic acid
present in the Folin Reagent results in blue purple color complex, with maximum absorption
46
in the region of 660 nm. The amount of colour produced is proportional to the amount of
peptide bonds, i.e. size as well as the amount of protein/peptide.
Materials:
Clean glass test tubes
Samples
Micropipettes
Cuvettes
Spectrophotometer
Reagents for Lowry’s Assay:
Reagent A:
2% Sodium Carbonate in 0.1 N Sodium hydroxide.
Reagent B:
0.5% Copper Sulphate in 1% Sodium Potassium Tartarate.
Reagent C:
50 ml of Reagent A was mixed with 1ml of Reagent B.
Reagent D:
Folin - Ciocalteau reagent:
Dilute commercial reagent (2N) with an equal volume of water on the day of use (2
ml of commercial reagent was diluted with equal volume of distilled water).
Preparation of BSA Standard Solution:
Stock Solution:
50 mg of BSA (Fraction V) was weighed and dissolved in distilled water and make
up to 50 ml in a standard measuring flask.
47
Working Solution:
10ml of stock solution was taken and make up to 50 ml with distilled water in a
standard measuring flask. 1ml of the solution now contains 200 µg protein.
Methodology:
In a series of test tubes, 20, 40, 60, 80 and 100 μl of the working standard solution
(Bovine serum albumin (Fraction V) (Hi-Media)) was added using a micro-pipette. In
another two test tubes 5 and 10 μl of the unknown protein sample was added similarly. All
the test tubes were made up into 1000 μl volume with sterile distilled water. A tube with 1ml
of water, serves as the blank. 5ml of Reagent C was added to all test tubes including the
blank. The tubes were mixed well and allowed to stand for 10 min. After incubation about
500 μl of reagent D was added and mixed well and incubated in the dark place at room
temperature for 30 min, the blue color was developed in the stipulated time period. Optical
density value was taken at 660nm in spectrophotometer. Standard graph was plotted against
BSA and the amounts of protein in the unknown samples were estimated.
3.9. SDS-PAGE:
Principle:
Electrophoresis is the study of the movement of charge molecules in an electric field.
Their rate of movement depends on the strength of the field, net charge, size and shape of the
molecules and also the ionic strength, viscosity and temperature of the medium in which the
molecules are moving. The generally used support medium is polyacrylamide.
Polyacrylamide gels are widely used for separating larger molecules like proteins. Proteins
are amphoteric in nature. Hence, the general electrophoresis techniques cannot be used to
measure the molecular weight of the protein molecules because the mobility of a substance in
the gel is influenced by both charge and size. In order to overcome this, if the protein
48
samples are treated so that they have a uniform charge, electrophoretic mobility that depends
primarily on size. The molecular weight of protein was estimated if they are subjected to
electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing
agent β-mercaptoethanol. SDS disrupts the secondary, tertiary and quaternary structure of the
protein to produce a linear polypeptide chain coated with negatively charged SDS molecules.
Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds (both interdisulfide and intra-disulfide bonds).
Materials:
Eppendorfs
Micro-pipettes
Micro-tips
Centrifuge
Water bath
Vortex
Distilled water
Chemical preparation of SDS-PAGE:
30% Acrylamide and 0.3% Bis acrylamide:
Acrylamide
- 30 g
N,N’ Methylene Bisacrylamide
- 0.3 g
Make up into 100 ml with distilled water.
4X Separating Buffer:
Tris HCl
Distilled water
- 18.2 g
- 60 ml
pH was adjusted to 8.8 with 10N NaOH
49
Sodium Dodecyl Sulphate (SDS) - 0.4 g
Make up into 100 ml with distilled water.
4X Stacking Buffer:
Tris HCl
- 6.05 g
Distilled water
- 40 ml
pH was adjusted to 6.8 with 10N NaOH
Sodium Dodecyl Sulphate (SDS)
- 0.4 g
Make upto 100 ml with distilled water.
2X Sample Buffer:
Tris stacking buffer (pH 6.8)
- 25 ml
Sodium Dodecyl Sulphate (SDS)
-4g
20% Glycerol
- 20 ml (should be added last and mixed well)
Make upto 100 ml with distilled water.
During protein sample preparation, 5% β-mercaptoethanol and during loading the protein
samples into the well, 5µl of Bromophenol blue was added.
10% Ammonium Per Sulphate (APS):
APS - 100 mg
Make upto 1 ml with distilled water.
1X Running Buffer:
Tris Hcl - 3.94 g
Glycine - 15.014 g
SDS
-1g
Make upto 1L with distilled water.
Staining Solution:
Coomassie Brilliant Blue R-250 – 200 mg
50
Methanol
- 50 ml
Glacial Acetic acid
- 7 ml
Make upto 100 ml with distilled water.
The above mixture was filtered using Whatman No 1 filter paper.
Destaining Solution:
Methanol
- 30 ml
Glacial Acetic acid
- 7 ml
Make upto 100 ml with distilled water.
Table 2: Separating gel concentration
Sl.
No
Stock
Solution
5%
6%
7%
7.5
%
8%
9%
10
%
12
%
13
%
15
%
1.
30% Acrylamide +
5.0
6.0
7.0
7.5
8.0
9.0
10.0
12.0
13.0
15.0
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
0.3% Bis-acrylamide (ml)
2.
4X Tris Separating
Buffer (ml) pH 8.8
3.
Distilled water (ml)
17.5
16.5
15.5
15.0
14.5
13.5
12.5
10.5
9.5
7.5
4.
10% APS (µl)
200
200
200
200
200
200
200
200
200
200
5.
TEMED (µl)
20
20
20
20
20
20
20
20
20
20
51
Table 3: Stacking gel concentration
Stock Solution
5 ml
10 ml
15 ml
20 ml
30% Acrylamide +
0.65
2.5
3.75
5.0
S
l.No
1.
0.3% Bis-acrylamide (ml)
2.
4X Tris Stacking Buffer (ml) pH 6.8
1.25
2.5
3.75
5.0
3.
Distilled water (ml)
3.05
5.0
7.5
10.0
4.
10% APS (µl)
100
100
100
100
5.
TEMED (µl)
10
10
10
10
Methodology:
Protein lysate preparation from different tissues of earthworm, E.eugeniae:
To gain an understanding of the protein profile of earthworm E.eugeniae, different
organs were dissected out and were resolved in the SDS-PAGE. The organ brain, testis,
ovary, skin, prostate, seminal vesicle was dissected and were initially washed in the 1X ice
cold PBS and aseptically transferred into a vial containing 2X protein sample preparation
buffer. The samples were uniformly homogenized by tissue homogenizer. The 2X
concentration was finally diluted to 1X with sterile water and were immediately transferred
into the boiling water bath for 10 minutes and then the protein sample was stored in -20ºC.
For the coelomic fluid sample the 100µl of isolated coelomic fluid was transferred to the
fresh vial and to that 100µl of 2X protein sample preparation buffer were added and mixed
together before heating the samples. The different protein samples were resolved in the 15%
gel along with protein markers purchased from Helini Biotech and were run at 100V for 8
52
hours. Proteins were stained with Coomassie Brilliant Blue – R250 staining and the results
were documented.
Protein lysate preparation from different tissues of earthworm, P.excavatus:
Inorder to understand the differences that are observed between the anterior and the
posterior part of the worm in the terms of regeneration. The tissue samples from an anterior
(1-10 segment) and posterior (25-35 segment) were subjected to proteomic analysis using
SDS-PAGE. The dissected samples were washed with
ice cold 1X PBS solution and
transferred to vial having 2X sample buffer and uniformly homogenized in ice cold
condition. The sample was diluted to 1X concentration with sterile distilled water before
heating in boiling water bath for 10 minutes. The processed samples were transferred to
attain the room temperature before storing the sample in -20ºC. The protein lysate prepared
were resolved in 15% SDS-PAGE gel.
Protein lysate preparation from normal and prostate cancer patient serum:
The normal and cancer patient’s serum are obtained from Neeiyoor international
cancer hospital with the permission and with the interest of the patient. From the separated
serum samples 100µl are transferred to a fresh vial and equal volume of the 2X serum sample
solution was added and mixed well prior to heating in boiling water bath for 10 minutes. The
protein lysate prepared from the serum samples were stored in -20ºC for future use.
Preparation of gel:
The glass plates were cleaned assembled so that the longer glass plate laid first, then 2
spacers of 1.5 mm thickness was placed along the sides of the rectangular plate. The notched
glass plate was placed on the top of the spacers so that the bottom ends of the spacers and
glass plates were aligned and the whole setup was assembled on the stand. 1% agarose was
prepared and it was used to seal the plates.
53
15% separating gel monomer solution (Table. 2) was prepared and carefully poured
into the glass plate assembly without forming air bubbles. The gel was overlaid with nbutanol so that it excludes the air. The separating gel was allowed to polymerase for 20 min.
The butanol layer was removed and the gel was washed with distilled water.
Stacking gel monomer solution (Table. 3) was then prepared and poured over the
separating gel. The comb was then inserted and the gel was allowed to solidify for 10 min.
After the gel gets solidified, the comb was removed carefully and the wells were washed with
distilled water. The assembled glass plate was then placed in the buffer chamber and running
buffer was poured in both upper and lower buffer chamber.
Sample loading and electrophoresis:
80 µg of sample and appropriate molecular markers were loaded in the wells. The
apparatus was connected to the power pack with proper polarity and 5 to 6 v/cm was applied.
After electrophoresis, the glass plate assembly was taken from the buffer chamber. The
spacer was gently twisted so that the upper glass plate pulls away from the gel.
Staining and destaining the gel:
The gel was carefully taken out from the glass plate and the stacking gel was
removed. Separating gel was removed by gently grasping the two corners of the gel and
placed in the container containing the commassie brilliant blue R-250. The gel should be
fully submerged in the staining solution for 2 hours by gentle agitation on a shaker. Then it
was washed with distilled water and transferred to a destaining solution until protein bands
are visualized clearly. The gel was then documented (Lark gel documentation system).
3.10. Gradient SDS-PAGE:
In order to separate the clump of same and closely related Molecular weight proteins,
manual Gradient SDS-PAGE gel was performed. The usual limits of Gradient level start
from 3% to 20%, but for the present experiment the gradient level starts from 6% to 15% to
54
resolve the target protein band. A similar-sized protein, but with slightly lower Molecular
weight were resolved to give a sharp band in the Gradient gel electrophoresis.
3.11. Collection of Blood samples:
The blood samples of normal patients were collected using the standard vinous
puncture. The cancer patient’s blood sample was drawn from the International Cancer
Center, Neyyoor, Kanyakumari district, South India.
3.12. PSA analysis of cancer patient serum:
PSA level is considered to be the most sensitive marker for monitoring patients with
known prostate cancer but it was not the absolute test for malignancy. The suspected prostate
cancer patient has the PSA value of 13.9ng/ml and the age and sex of the patient is a 66 year
male. Following the PSA value the prostate cancer of the patient were also confirmed by
biopsy.
3.13. Serum sample preparation:
Immediately after drawing the blood from the normal and cancer patients it was
transferred to the 15ml tube and kept undisturbed for 30 to 45 minutes. After the blood got
coagulate it was centrifuged at 4,000 rpm for 20 minutes. After centrifugation the clear
yellow color fluid of serum was carefully separated from the supernatant. The collected
serum samples from normal and cancer patients were stored at -20ºC.
3.14. Mass spectrometry analysis:
The interested protein bands 23kDa size of the coelomic fluid and the prostate gland
of E.eugeniae and 20kDa size from the post clitellum segments of E.eugeniae was sliced out
from the SDS – PAGE gel and stored in 1% acetic acid in a sterile vial and they were sent for
Mass spectrometry analysis to a German institute. The mass spectrometry results show 116
55
peptide sequences for prostate protein and 92 peptide sequences for coelomic fluid protein
each peptides range from 9 - 25 amino acids. The mass spectrometry results for 20kDa size
protein shows 97 peptide sequences each peptides range from 9 - 29 amino acids. Global
alignment of the entire peptide sequence was performed using NCBI protein blast to find out
the prostate related protein. General comparisons between the distribution of prostate related
protein in different organism were analyzed using Align X software to reveal the similarity
between the different species. The identified protein for 20kDa size was further aligned from
higher to lower organisms using the Align-X software.
3.15. Immunoblotting:
The overnight run SDS-PAGE gel was transferred to 1X TBST buffer and incubates
for 15 minutes. The gel size cutted PVDF membrane was activated by dipping in methanol
and before it dry, it is transferred to 1X TBST buffer. The gel size cutted whatman filter
paper (soaked in 1X TBST buffer) was placed one by one on the transferring unit. The PVDF
membrane was placed over the filter paper and then the gel was carefully placed on the
PVDF membrane. The whatman filter paper was again stacked over it. The transferring unit
was set to 1.8volts for 20 minutes to allow the transfer of protein bands from the gel to the
PVDF membrane. The transferred PVDF membrane was then blocked with 4% skimmed
milk to prevent non-specific binding of the antibody and latter incubated with primary
antibody (dilution 1:500) for overnight at 4ºC. The nonspecific binding of 1º Ab was then
washed out with a 1X TBST buffer. The 2º Ab conjugated with Alkaline phosphatase was
used with the dilution concentration of 1:3000. The washed membrane was developed with
the substrate BCIP-NBT solution to get the blue colored product.
56
3.16. Sample and specimen preparation for fluorescence microscopic analysis:
3.16.1. Fixation of earthworm:
The setae of the earthworm, E.eugeniae and P.excavatus has the auto fluorescent
property. Inorder to study the setae arrangement in between these two worms, the worms
were initially fixed with 10% formalin solution for 5 minutes and examined under the
fluorescent microscope in blue, green and red filters. Inorder to study the regeneration ability
of setae the earthworm, E.eugeniae and P.excavatus were amputated at the pre-clitellum and
within the clitellum segments. The 3rd to 8th day bud were fixed initially with 10% formalin
solution for 5 minutes and examined under the fluorescent microscope for the identification
of setae formation.
3.16.2. Isolation of the earthworm setae:
The earthworm was washed with running water to remove any debris or dust from the
body segment. The earthworm was cut into five segments and they are dissected dorsally to
remove any dirty and unwanted tissue. The dissected tissue was carefully placed on the slide
and a few drops of 10% KOH solution was placed over it and incubates for 2 hours. After
incubation rinsed repeatedly, transferred into a watch glass with some distilled water and
picked out manually under a fluorescent microscope.
3.16.3. Autofluorescence ability of tissue sections in earthworm, E.eugeniae and
P.excavatus:
In order to study the autofluorescence ability in the worm E.eugeniae and P.
excavatus, a 7µm thin section of earthworm tissue was subjected to florescent microscopic
analysis. The 7 micron thin histological tissue sections were initially placed in egg
albumenized slides (egg white was mixed with equal volume of glycerol and spread over the
slides). The slides were placed in slide warmer and the temperature was maintained at 60οC.
57
Little amount of water was sprinkled over the slides and the thin sections were placed in the
slide containing water. As the slide is on the slide warmer, the wax get melts at that time the
water was removed and the slide was again placed in the slide warmer for about 10 min.
Then the slides were dewaxed by dipping in xylene for 5 min followed by dipping in 100%
isopropyl alcohol for 3 min, mounted with DPX mounting agent and viewed under a Nikon
Ti-S inverted fluorescent microscope for autofluorescence without any staining pattern, after
which the slides were stained with a haematoxylin - Eosin combination for further
documented.
3.16.4. Autofluorescence in coelomic fluid cells:
To observe the autofluorescence of coelomic fluid cells, coelomic fluid was collected
from the mature worm by giving 4.5 volts. Due to electric shock, the earthworms were start
to releasing the ceolomic fluid, it was collected in the eppendorf tube and smeared on a clean
glass slide. The smear was fixed with 4% Paraformaldehyde and they are briefly washed with
1X PBS; and it was examined under a Nikon Ti-S inverted fluorescence microscope.
3.16.5. Spectroflourimetry analysis:
Spectrofluorometric analyses were performed using the lysate of earthworm.
Earthworm tissues were homogenized with sterile distilled water and the lysate obtained was
centrifuged at 5000 rpm for 10 min (Eppendorf Mini Spin Centrifuge). The supernatant was
collected, and it was subjected to spectrofluorometric analyses with standard riboflavin,
FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide) (purchased from
HiMedia Laboratories Private Limited, India).
Spectrofluorometry analysis was performed using the lysate of isolated setae. The
setae was isolated by using KOH treatment and isolated setae were subjected to
58
spectrofluorometry analysis with standard riboflavin, FMN (flavin mononucleotide) and
FAD (flavin adenine dinucleotide) solution.
3.17. BrdU labeling retention assay:
Principle:
Bromo-deoxy-uridine (5-bromo-2'-deoxy-uridine, BrdU - purchased from Sisco
Research Laboratories Private Limited, India) is an analog of the DNA precursor
‘Thymidine’. Injection of BrdU into the earthworm, labels all the cells (including the adult
stem cells). After a prolonged period of incubation (commonly known as chasing) the BrdU
concentrations in all proliferating cells became reduced and sloughed off. But, the
incorporated BrdU in the adult stem cells became retained, because stem cells have a slow
proliferation rate other than normal cells. They can divide only if they have a tendency to
renew or to restore the damaged organs or body segments. The amount of BrdU in the DNA
of cells can be detected with specific monoclonal anti-BrdU antibody. Thus, labeling
retention assay is used to identify the location of stem cells and its niche.
Materials:
Screw cap bottles
Clean Albumenized glass slides
Coupling jars
Cover slips
BrdU stock solution:
BrdU stock solution (10 µg/ml)
10 mg of BrdU was dissolved in 1 ml of 1X PBS.
1 µl of stock solution contain 10 µg of BrdU
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Preparation of 10X PBS:
NaCl
– 80.0 g
KCl
– 2.0 g
Na2HPO4
– 14.4 g
K2HPO4
Distilled water
– 2.4 g
– 800 ml
pH was adjusted to 7.4 with HCl
The solution was make up into 1000 ml with distilled water, autoclaved and stored at
room temperature.
1X PBS:
100 ml of 10X PBS and makes up into 1000 ml with sterile distilled water
TBST:
When Tween 20 was added to PBS it was called as TBST
For 1000 ml, 1 ml of Tween 20 was added to 1X PBS
Fixation:
10% Formaldehyde
Dehydration:
Different grades of isopropyl alcohol were used for dehydrating the tissues as follows:
60% isopropyl alcohol
70% isopropyl alcohol
80% isopropyl alcohol
90% isopropyl alcohol
100% isopropyl alcohol
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100% isopropyl alcohol
Clearing:
Xylene
Impregnation with wax:
Paraffin wax – 1
Paraffin wax – 2
Paraffin wax – 3
Dewaxing:
Xylene
Dehydration:
100% isopropyl alcohol
Permeabilization:
0.25% Triton X 100
Endogenous peroxidase blocking:
H2O2
- 10 ml
Methanol - 10 ml
Make upto 100 ml with 1X PBS
Denaturation:
2N HCl
Blocking:
2% Bovine Serum Albumin in TBST.
Primary Antibody:
Mouse monoclonal anti-BrdU antibody 1: 200 (from Sigma)
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Secondary antibody:
Goat anti mouse IgG HRP 1:500
Developing using Diaminobenzidine (DAB) kit (for 2ml):
Dilution buffer - 2 ml
H2O2
- 2 µl
DAB
- 40 µl
Counter stain:
Erhlich Haematoxylin
Acid alcohol:
1ml of concentrated HCl was mixed with 99ml of 70% isopropyl alcohol.
Decolourizer:
100% isopropyl alcohol
Methodology:
The BrdU stock solution was prepared at a concentration of 10mg/ml in 1X PBS and
each worm was injected with 10µl of BrdU from the 20th segment to the 24th segments. The
worm injected with BrdU was maintained for 30 days for chasing. On the 30th day the worm
was amputated at the 11th segment and allowed to regenerate the lost part. The regeneration
blastema appeared on the 4th day, and it was formalin-fixed, paraffin-embedded, sectioned
and allowed to stain with anti-BrdU antibody.
3.18. Immunohistochemistry:
To visualize BrdU incorporation, paraffin-embedded earthworm tissue sections (6µm)
were de-paraffinized with xylene and hydrated. Endogenous peroxidase was inhibited by
incubation of the sections for 30 min with freshly prepared 10% H2O2 and 10% Methanol in
1X PBS. The sections were then treated with 0.1% Trypsin in 0.1% CaCl2 at 37°C for 10
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min. DNA was denatured by incubating the section with 2N HCL at 37°C for 45 min.
Nonspecific staining was blocked by treatment with 2% BSA for 1 hour at ambient
temperature. The sections were then incubated overnight at 4oC with mouse monoclonal antiBrdU antibody (Clone BU-33, Sigma) at a dilution of 1:500 in BSA. After incubation with
primary antibody, tissue sections were washed and incubated for 1 hour with goat anti-mouse
IgG conjugated with horseradish peroxidase (Lot No: 062100; GeNei) at a dilution of
1:1000. Staining was developed with Diaminobenzidine (DAB Kit; GeNei) substrate and the
sections were counterstained with Ehrlich haematoxylin. The prepared slides were mounted
with DPX and observed under a Nikon Ti-S microscope.
3.19. Histochemistry for Alkaline Phosphatase:
The regenerated blastema of E.eugeniae were fixed with 100% methanol for 15 min
and washed with 1X TBST and stained with BCIP/NBT solution (from amresco) (which is a
substrate for alkaline phosphatase) for 90 min at room temperature. After the incubation in
the BCIP/NBT solution, the worm tissues were washed with 1X TBST, fixed with AGE
fixative (Acetic acid (4): Glycerol (1): Ethanol (2)) for 15 min and whole mounted with
DPX. The prepared slides were observed under a Nikon Ti-S microscope for Alkaline
Phosphatase (ALP) activity.
3.20. Counting of cells:
The BrdU positive cells stained with anti-BrdU antibody and developed with DAB
shows dark brown colour nucleus and the other cells were stained with haematoxylin. The
slides were carefully observed under the Nikon Ti-S microscope and the BrdU positive cells
were counted. The counting was repeated thrice to verify the data and also for the statistical
analysis. Similarly, autofluorescent cell has been counted under the Nikon Ti-S fluorescent
microscope.
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3.21. Statistical analysis:
Statistical analysis such as standard deviation, p-value for BrdU-positive cells versus
a positive control, and difference in the flurocent intensity between the coelomic fluid of
E.eugeniae and P.excavatus were performed using Microsoft Office Excel 2007.
3.22. Generation of Polyclonal antibody against the 20kDa size protein of E.eugeniae:
For raising the polyclonal antibody the animal system selected was a rabbit model.
The female rabbit of four months old is a better choice because it is more active than a male
rabbit. The desired protein of interest was sliced out from the gel and they are homogenized
using sterile 1X PBS buffer solution. The 300µl of the homogenized was injected into the
rabbit system subcutaneously. The rabbit is fed with green leaves like cabbage and dry pellets. If
the animal is found to be very week give carrots or else no need to change the food. Place
sufficient drinking water (human drinking grade) inside the cage in a bowl. After 20 days of
primary dosage, the subsequent secondary dose was given (exactly on 21th day) and the rabbit is
carefully monitored. After 10 days of secondary dosage the rabbit was sacrificed to draw the
blood. The obtained blood sample after 30 minutes coagulation they are subjected to centrifuge
to get the clear pale yellow coloured serum sample. Now the serum has the antibody against the
20kDa size protein of E.eugeniae and the raised antibody is cross checked using the Western blot
analysis.
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