GENERAL MATERIALS AND METHODS

Animals

The animal model used in this study is laboratory mouse, Mus musculus of

Swiss strain. These mice were bred either in the animal house of the Department of

Zoology, Institute of Science or Institute of Medical Sciences, Banaras Hindu

University, Varanasi. The colony is closed and randomly bred.

Phylum : Chordata
Class : Mammalia
Order : Rodentia
Family : Muridae
Genus : Mus
Species : musculus
Fig. F: Laboratory mouse, Mus

musculus

Mice were maintained under hygienic conditions in a photoperiodically

controlled (LD 12:12) and well ventilated room (25 3oC). 5-6 mice were kept

together in a polypropylene cages (430mm x 270mm x 150mm) with dry rice husk as

the bedding material and supplied with standard rodent food pellets (supplied by

Pashu Aahar Kendra, Varanasi) and tap water ad libitum. All the experimental studies

were conducted in accordance with institutional practice and within the framework of

the revised Animals (Scientific Procedures) Act of 2002 of the Government of India.

Methodology

Specific study design and protocol for each experiment is described separately

in respective sections. This description pertains to general methodology used for all

the studies. At the termination of each experiment, animals were sacrificed either by
 General Materials & Methods

decapitation or following ether anaesthesia. Blood was collected into tubes and

centrifuged at 4000 rpm for 20 min at 40C to separate the serum for the ELISA of

corticosterone. For immunohistochemistry/ immunofluorescence ether anaesthetised

mice were processed for whole body perfusion. Serum and tissues of mice sacrificed

by decapitation were used for biochemical estimations and RNA extraction.

Whole body perfusion

After the completion of each experiment some mice were perfused

transcardially (through left ventricle) with PBS followed by 4% paraformaldehyde

(PFA) in 0.02M PBS (pH 7.4). Brain was separated and post-fixed in 4% PFA for 18-

20 hrs and then transferred to 70% ethyl alcohol for storage.

Homogenate preparation

For the antioxidant enzyme activity assay and nitric oxide measurement,

hypothalamus homogenate was used. Hypothalamus was separated and 10%

homogenate (w/v) was prepared in chilled PBS (0.02M, pH 7.4) using electrical

homogenizer in the cold room. This homogenate was centrifuged at 12000 rpm at 4 0C

for 20 min. Supernatant was collected and required amount was immediately used for

biochemical estimation of nitric oxide level and rest was stored at -20 0C for the

antioxidant enzyme activity assay.

Antioxidant enzyme activity assay

Superoxide dismutase activity (SOD)

SOD activity was assayed by the method of Das et al. (2000) in the

hypothalamus. The reaction mixture contained 100 l of sample, 150 mM phosphate

buffer (pH 7.4), 20 mM L-methionine, 50M EDTA (Ethylenediaminetetraacetic

26
 General Materials & Methods

acid), 1 % Triton X-100, 100 M riboflavin, and Greiss reagent. The absorbance was

taken at 543 nm. The value of SOD was calculated in terms of units defined as the

amount of SOD that inhibits the reduction of nitroblue tetrazolium (NBT) by 50%.

The final results were expressed as unit of SOD per mg of protein.

Catalase assay (CAT)

Catalase activity in the hypothalamus was assayed according to Aebi (1984).

Briefly, the reaction mixture contained 1.9 ml of 50 mM phosphate buffer (pH7.0) and

appropriate dilution of the tissue supernatant to make the volume 2 ml. The reaction

was initiated by the addition of 1 ml of freshly prepared 30 mM H 2O2 and the

decrease in absorbance was measured at 240 nm for 23 min. One unit of catalase

represents the decrease of 1 M of H2O2 per minute. The molar extinction coefficient

for H2O2 is 43.6 M1cm1.

Biochemical estimation of nitric oxide (NO) level

NO was determined spectrophotometrically by measuring the accumulation of

its stable products in the form of nitrate-nitrite. Total nitrate-nitrite concentration was

measured in mouse hypothalamus according to Sastry et al. (2002). Briefly, 10 %

tissue homogenate (w/v) was prepared in 0.01M phosphate buffer pH 7.4. To 100 l

of tissue sample or standard, 400 l of carbonate buffer was added followed by a

small amount (0.15 g) of activated coppercadmium alloy fillings and incubated at

room temperature with thorough shaking. The reaction was stopped by the addition of

100 l of 0.35M NaOH followed by 120mM ZnSO4 solution under vortex and

allowed to stand for 10 min. Tubes were then centrifuged at 8000 rpm for 10 min. 100

l of aliquots of clear supernatant were transferred into the 96 well micro plate (in

27
 General Materials & Methods

triplicate) and Griess reagent (50 l of 1% sulphonilamide prepared in 2.5%

orthophosphoric acid and 50 l of 0.1% N-naphthyl ethylenediamine (NEDD)

prepared in distilled H2O) was added to it. After 10 min, the absorbance was read at

545 nm in Micro Scan (MS56084, Electronics Corporation of India Limited). A

standard graph was plotted against different concentrations (0, 20, 40, 60, 80 and

100M) of KNO3. Results were expressed as nmoles/mg of protein.

RNA Extraction

The total RNA from hypothalamus and testis of the mice of various groups

were extracted using TRIzol Reagent (Ambion, life technologies) according to its user

manual, dissolved in DEPC - treated water and quantified using spectrophotometer.

cDNA synthesis

cDNA was synthesized from 10 g of total RNA using random hexamer

primer and reverse transcriptase. In brief, 10 g of RNA was mixed with 200 ng of

random hexamer in DEPC treated water in the reaction volume of 11 l. It was

incubated at 70oC for 5 min. Thereafter the following components were added in the

order indicated: 5 x reaction buffer-4.0 l, dNTP mix (2.5mM each) - 2.0 l, RNase

inhibitor - 0.5 l, DEPC treated water - 1.5 l. The tubes containing reaction mixture

were incubated for 5 min at 25oC and 1.0 l of M-MuLV reverse transcriptase

(RevertAid, 200 units) was added. Further, the tubes were incubated for 10 min at

25oC and then at 42oC for 1 h. The reaction was terminated by heating at 70 oC for 10

min, and after chilling, the tubes were stored at -20oC to be used directly for the PCR

reaction.

28
 General Materials & Methods

Polymerase chain reaction

Specific primers (forward and reverse) were used for the analysis of specific

genes (Table A). Specific oligonucleotides (primers) used for the analysis of AVP,

CRH, nNOS, NF-B, GST M1, GST P1 and -actin were synthesized by mwg -

operon, Bangalore, India. The PCR was performed in 25 l reaction mixture

containing 2 l cDNA, 10x Taq polymerase buffer, 2.5 mM of each dNTP

(Biobharati, India), 1.0 unit of Taq DNA polymerase (Biobharati, India), and 10 pmol

of appropriate specific primers (forward and reverse). The reactions were carried out

in T 100TM BioRad Thermal cycler. The samples were denatured at 94oC for 5 min and

amplified using different conditions for different genes (Table B).

Table A. Sequences of primers for the specific genes used in RT-PCR

Gene Primer sequence Product

size
actin Forward 5- ATC GTG GGC CGC TCT AGG CAC C - 3 543 bp
Reverse 5- CTC TTT GAT GTC ACG CAC GAT TTC- 3
AVP Forward 5- ACT ATG CAC GAC TTC GGG TG- 3 205 bp
Reverse 5- AGT CCG TGG ATT CTG CCA AG- 3
CRH Forward 5- GTA CAG AGG AAA GCC CAG GAC- 3 337 bp
Reverse 5- TTC TTG AGG GGT GGC TAG GA 3
nNOS Forward 5- CCT GGG GCT CAA ATG GTA TG - 3 373 bp
Reverse 5- CAC AAT CCA CAC CCA GTC GG - 3
NF-B Forward 5- TAG AAT GAG CTG TGG GCT TGA - 3 518 bp
Reverse 5- ACC GCA TGA TCA ACC CAT CC - 3
GST Forward 5- CCTATGATACTGGGATACTGGAACG- 3 113 bp
M1 Reverse 5- GGAGCGTCACCCATGGTG- 3
GST P1 Forward 5- GCAAATATGTCACCCTCATCTACACC-3 96 bp
Reverse 5- GCAGGGTCTCAAAAGGCTTCA- 3

Table B. Amplification conditions for different genes

29
 General Materials & Methods

Gene Denaturation Primer annealing Elongation No. of cycles

actin 94oC - 30 sec 57oC 30 sec 72oC 30 sec 30
AVP 94oC - 45 sec 62oC 45 sec 72oC 1 min 35
CRH 94oC - 45 sec 64oC 40 sec 72oC 1 min 35
nNOS 94oC 40 sec 65oC 40 sec 72oC 1 min 32
NF-B 94oC 40 sec 62oC 40 sec 72oC 1 min 35
GST M1 94oC 1 min 61oC 1 min 72oC 1 min 35
GST P1 94oC 1 min 61oC 50 sec 72oC 1 min 35

The resultant PCR products were electrophoresed on 2% agarose gel

containing ethidium bromide, bands were visualized using UVtransilluminator

Alpha Digi doc from Alpha Innotech (India) and photographed by Olympus camera

(USA). The densitometric analysis of band intensity was done by ImageJ software

and normalized by -actin. The expression of AVP, CRH, nNOS, NF-B, GST M1

and GST P1 genes were expressed as percent (%) band intensity relative to that of

actin.

Immunohistochemistry (IHC)

Following whole body perfusion brains were excised and fixed for 24 hrs in

4% (w/v) paraformaldehyde, dehydrated and embedded in paraffin. Tissue blocks

were prepared and 8 m thick coronal sections of brain were cut on a microtome

(LEICA) and collected on gelatin- coated slides. Immunohistochemistry was

performed in the brain sections for different peptides as described by Singh and

Chaturvedi (2014), with some modifications. In brief, the brain sections were

deparaffinized, rehydrated in graded series of ethanol and rinsed in PBS and then

immersed in 0.3% hydrogen peroxide for 20 min at room temperature. They were

preincubated in 10% normal goat serum for 1 hr and subsequently incubated in

specific primary antibody for overnight at 4oC. The second incubation with Vectastain

30
 General Materials & Methods

Avidin-Biotin complex Kit and 20 antibody (Vector Laboratories, Burlingame, CA,

USA), were visualized with 0.025% 3, 3-diaminobenzidine (Sigma) in PBS

containing 0.02% H2O2. The slides were washed with PBS, dehydrated through graded

series of ethanol, cleaned in xylene and mounted using DPX. Slides were viewed

under Carl Zeiss Axioskop 2 Plus microscope.

Details (source and dilution) of the primary antibodies used for

immunohistochemistry or immunofluorescence are presented in Table C.

Table C. Antibodies and their dilution used for immunohistochemistry/

immunofluorescence

Primary Source Dilution used

antibody
AVP Rabbit, Merck Millipore, Germany 1:1000
CRH Rabbit, Santa Cruz Biotechnology, USA 1:50
nNOS Rabbit, Santa Cruz Biotechnology, USA 1:50
Hsp70 Rat, gifted by Dr. S.C. Lakhotia, Varanasi, India 1:100

Immunofluorescence (IF)

For the immunofluorescence brain sections were deparaffinized, rehydrated in

graded series of ethanol, rinsed with PBS and incubated in blocking solution (1%

BSA, NGS and Tween20) for 2 hrs. Sections were incubated in 0.03% H2O2 for 30

min subsequently incubated in specific primary antibody for overnight at 4oC, washed

in PBST for 10 min. The antigens were visualized using appropriate secondary

antibody conjugated with Fluorescein FITC (Anti-rabbit, 1:200; Keyman Chemical

Company) or Rhodamine (TRITC Anti-rat, 1:200) for 1 hour at room temperature;

the slides were rinsed in PBS. Nuclei were stained with 4, 6- diamino-2-phenylindole

(DAPI) and were analysed for fluorescence under fluorescence microscope (Carl

Zeiss Axioskop 2 Plus).

31
 General Materials & Methods

Although, all the antibodies used in the present study are specific and has

already been used and reported earlier (by our lab as well as others workers), even

then negative controls were processed for confirming the specificity of each antibody.

For negative control, of IHC instead of primary antibody, sections were incubated

with PBS only. Rest of the procedure i. e. secondary antibody and subsequent washing

etc. was same. Microphotograph of one of the negative control (representative) slide

is shown in Fig. G.

Fig. G: Coronal brain section passing through the hypothalamus of mouse showing
negative control for immunohistochemistry (3V= 3rd ventricle)

ELISA

Serum corticosterone level was measured by using ELISA kit (Abcam

ab108821, UK) according to the manufacturers standard protocol provided with the

kit. Absorbance was taken at 450 nm in Micro Scan (MS56084, Electronics

Corporation of India Limited).

Statistical analysis

The gels were photographed by a digital camera (Olympus) and the

densitometric analysis of bands was done by ImageJ software. For the RT PCR,

32
 General Materials & Methods

band intensity of AVP/ CRH was measured and normalized with actin and expressed

as % relative expression. The photographs of brain IHC and immunofluorescent slides

were taken from Carl Zeiss Axioskop 2 Plus microscope. All the data were analysed

either by one - way ANOVA followed by Tukeys test or by Students t test for the

comparison of groups from control. Difference was considered significant at the level

of P<0.05.

*******

33