Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Breast cancer tissue and adjacent normal)
Specification
Breast cancer tissue and adjacent normal
Fixative
Formaldehyde
Antigen retrieval step
Other
Permeabilization
No
Blocking step
H2o2 of abcam secondary antibody Kit Expose Kit as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 37°C
Other product details
Dilution
1/50
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 37°C · Diluent: TBS as given by abcam protocol
Secondary antibody
Name
Abcam antibody:EXPOSE Rabbit specific HRP/DAB detection IHC kit
Additional data
Additional Notes
This Abreview is submitting by ALLIED SCIENTIFIC PRODUCTS,INDIA On Behalf of our customer Dr Syeda Zubeda, Hyderabad
Solutions and Reagents:
a) Preparation of Coated Slides:
Table 15: Reagents for coating slides
Chemical Amount
Gelatin 4g
Chromalum 5g
Dist. H2O 1 lit
Gelatin and chromalum was dissolved in water by heating. The chemicals were let to dissolve completely.
Slides were coated with gelatin-chromalum solution: slides were dipped 3 times with a gap of 2 mins each after air drying.
b) Deparaffinization and Rehydration of tissue sections using a xylene and ethanol series:
Table 16: Deparaffinization and rehydration reagents
Chemical Amount
Xylene 250ml
100% Ethanol 250ml
95% Ethanol 250ml
70% Ethanol 250ml
Dist. H2O 250ml
c) Antigen retrieval method: -Sodium Citrate Antigen Retrieval:
Reagents:
Table 17: Sodium citrate Buffer 0.01M, pH 6.0:
Chemical Amount
Citric acid monohydrate 10.51g
Trisodium citrate dehydrate 14.7g
Dist. H2O 1 lit
Solution A. (0.1 M citric acid solution): 10.51gm of citric acid monohydrate (C6H8O7.H2O) was dissolved in 500ml of dH2O.
Solution B. (0.1M sodium citrate solution): 14.7 gm of trisodium citrate dehydrate (C6H5Na3O7.2H2O) was dissolved in 500ml of dH2O.
Working solution: 9ml of solution A and 41 ml of solution B was added to 450ml of dH2O. pH adjusted to 6.0.
d) Phosphate Buffer Saline: (PBS)
Reagents:
Table 18: Phosphate Buffer Saline composition
Chemical Amount
diSodium hydrogen orthophosphate 10.51g
Sodium hydrogen orthophosphate 14.7g
Sodium chloride 4.5g
Solution A: 10.6 g diSodium hydrogen orthophosphate (Na2HPO4) in 500ml of dH2O.
Solution B: 11.7 g of Sodium hydrogen orthophosphate (Na HPO4) in 500ml of dH2O.
Solution C: 4.5 g of Sodium chloride (NaCl) in 500ml of dH2O.
Working solution: 68ml of solution A, 32ml of Solution B and 100ml of Solution C.
e) Primary Antibody:
Reagents:
Table 19: Details of Primary antibody
Primary Antibody Company Catalog No.
Anti- AIF ABCAM, USA Ab-1998
Table 20: DAB reagents
Primary Antibody Volume
DAB substrate 1 ml
DAB chromogen 20 ul
g) Counterstaining:
Table 21: Staining solution reagents
Chemical
Hematoxalin QS
Acid
Ethanol
Dist. H2O
Tap H2O
h) Dehydrate sections:
Table 22: Reagents for Dehydration
Chemical Amount
95% ethanol 250ml
100% ethanol 250ml
Xylene 250ml
Protocol:
1. 4 um thick tissue sections were cut from paraffin blocks using microtome and
transferred into a 48oC water bath for the adherence of the tissue to the pre-
coated slide.
2. Slides with the sections were subsequently air dried at 37 oC and stored in box at
room temperature until further processing .
3. Tissue sections were baked in oven at 68 oC for 30 minutes prior to IHC staining.
4. Slides were immersed immediately in Xylene for 5 minutes each with 2 changes
of xylene followed by dipping sequentially in 100%, 90% and 70% ethanol.
5. Slides were placed in running water for 5 minutes and then washed in distilled
water.
6. Slides were placed in a glass slide holder and any empty slots in the rack were
filled with blank slides to ensure even heating before placing in 600ml of 10mM
sodium citrate working buffer. A line was marked at the top of the liquid on the
glass bowl.
7. The bowl was microwave for 20 minutes (4 cycles of 5 minutes each), the
evaporated buffer was replaced after 10 minutes
8. Slides were cooled for 20 minutes in the beaker, washed with dist.water for 5
minutes. Cleaned with the tissue paper without touching the sections.
9. Sections were circled with a glass marker. A drop of peroxidase block (blocking
buffer) was added to each section immediately, so that the sections do not dry out
(Do not touch sections with tip).This was kept as such for 10 minutes.
10. Slowly tilt the slide on a tissue paper so as to remove the peroxidase. Wash with
a drop of 1X PBS twice for 5 minutes each. Clean the slide with tissue paper.
Add blocking solution (Protein block) and keep for 10 minutes.
11. Remove the blocking solution and add 100 ul of reconstituted primary antibody (1:50
dilution) per section.
12. Incubated for 1 hour at room temperature in a humidified chamber not allowing
the slides to touch each other.
13. Primary antibody was drained off the section and slides were flooded for 2 times
in 1 X PBS for 5 minutes each.
14. Add Secondary antibody conjugate onto the sections and keep for 15 minutes
at room temperature .
17. Secondary antibody was drained off and slides were washed with 1XPBS twice for 5 minutes each.
18. 100 ul of DAB mixture (1 ml of DAB substrate and 20 ul of DAB chromogen is added ) and incubated in dark for 5 min. Then the sections are given two washes with 1XPBS for 5 min each.
19. Dipped in Hematoxalin for a minute
20. Rinsed with Dist. Water
21. Kept under Tap water for 5 minutes(to allow stain to develop)
21. Counterstaining:Slides were dipped in 90% ethanol 3 times for a period of
5 minutes. Followed by 100% ethanol 3 times for 5 minutes. Slides were then
dipped in Xylene 2 times for a period of 5 minutes each.
22. Mounting:A drop of Permount was placed on the slide using a glass rod. The
coverslip was gently placed on to the slide to allow the Permount to spread
beneath the coverslip, covering the whole tissue.
23. Slides were dried overnight in the hood.
Analysis:
Slides were then analyzed for AIF expression. Semi quantitative scoring was performed
at a magnification of X400. From each section, five regions were captured using charge coupled device camera attached to Nikon E400 microscope (Japan). Expression was scored on the basis of intensity as follows: 0, absent or no expression 0.5, mild expression 1, moderate expression 2, high expression and 3, intense expression. Two independent, unbiased scorers who were blinded to the origin of the histological specimen performed the analysis. Different sections from each tumor and normal sample were scored, and a mean of that was taken for analysis. Any discrepancies in scores were resolved by discussion with a pathologist.
NORMAL TUMOR
AIF : Abcam 1998 polyclonal antibody
Solutions and Reagents:
a) Preparation of Coated Slides:
Table 15: Reagents for coating slides
Chemical Amount
Gelatin 4g
Chromalum 5g
Dist. H2O 1 lit
Gelatin and chromalum was dissolved in water by heating. The chemicals were let to dissolve completely.
Slides were coated with gelatin-chromalum solution: slides were dipped 3 times with a gap of 2 mins each after air drying.
b) Deparaffinization and Rehydration of tissue sections using a xylene and ethanol series:
Table 16: Deparaffinization and rehydration reagents
Chemical Amount
Xylene 250ml
100% Ethanol 250ml
95% Ethanol 250ml
70% Ethanol 250ml
Dist. H2O 250ml
c) Antigen retrieval method: -Sodium Citrate Antigen Retrieval:
Reagents:
Table 17: Sodium citrate Buffer 0.01M, pH 6.0:
Chemical Amount
Citric acid monohydrate 10.51g
Trisodium citrate dehydrate 14.7g
Dist. H2O 1 lit
Solution A. (0.1 M citric acid solution): 10.51gm of citric acid monohydrate (C6H8O7.H2O) was dissolved in 500ml of dH2O.
Solution B. (0.1M sodium citrate solution): 14.7 gm of trisodium citrate dehydrate (C6H5Na3O7.2H2O) was dissolved in 500ml of dH2O.
Working solution: 9ml of solution A and 41 ml of solution B was added to 450ml of dH2O. pH adjusted to 6.0.
d) Phosphate Buffer Saline: (PBS)
Reagents:
Table 18: Phosphate Buffer Saline composition
Chemical Amount
diSodium hydrogen orthophosphate 10.51g
Sodium hydrogen orthophosphate 14.7g
Sodium chloride 4.5g
Solution A: 10.6 g diSodium hydrogen orthophosphate (Na2HPO4) in 500ml of dH2O.
Solution B: 11.7 g of Sodium hydrogen orthophosphate (Na HPO4) in 500ml of dH2O.
Solution C: 4.5 g of Sodium chloride (NaCl) in 500ml of dH2O.
Working solution: 68ml of solution A, 32ml of Solution B and 100ml of Solution C.
e) Primary Antibody:
Reagents:
Table 19: Details of Primary antibody
Primary Antibody Company Catalog No.
Anti- AIF ABCAM, USA Ab-1998
Table 20: DAB reagents
Primary Antibody Volume
DAB substrate 1 ml
DAB chromogen 20 ul
g) Counterstaining:
Table 21: Staining solution reagents
Chemical
Hematoxalin QS
Acid
Ethanol
Dist. H2O
Tap H2O
h) Dehydrate sections:
Table 22: Reagents for Dehydration
Chemical Amount
95% ethanol 250ml
100% ethanol 250ml
Xylene 250ml
Protocol:
1. 4 um thick tissue sections were cut from paraffin blocks using microtome and
transferred into a 48oC water bath for the adherence of the tissue to the pre-
coated slide.
2. Slides with the sections were subsequently air dried at 37 oC and stored in box at
room temperature until further processing .
3. Tissue sections were baked in oven at 68 oC for 30 minutes prior to IHC staining.
4. Slides were immersed immediately in Xylene for 5 minutes each with 2 changes
of xylene followed by dipping sequentially in 100%, 90% and 70% ethanol.
5. Slides were placed in running water for 5 minutes and then washed in distilled
water.
6. Slides were placed in a glass slide holder and any empty slots in the rack were
filled with blank slides to ensure even heating before placing in 600ml of 10mM
sodium citrate working buffer. A line was marked at the top of the liquid on the
glass bowl.
7. The bowl was microwave for 20 minutes (4 cycles of 5 minutes each), the
evaporated buffer was replaced after 10 minutes
8. Slides were cooled for 20 minutes in the beaker, washed with dist.water for 5
minutes. Cleaned with the tissue paper without touching the sections.
9. Sections were circled with a glass marker. A drop of peroxidase block (blocking
buffer) was added to each section immediately, so that the sections do not dry out
(Do not touch sections with tip).This was kept as such for 10 minutes.
10. Slowly tilt the slide on a tissue paper so as to remove the peroxidase. Wash with
a drop of 1X PBS twice for 5 minutes each. Clean the slide with tissue paper.
Add blocking solution (Protein block) and keep for 10 minutes.
11. Remove the blocking solution and add 100 ul of reconstituted primary antibody (1:50
dilution) per section.
12. Incubated for 1 hour at room temperature in a humidified chamber not allowing
the slides to touch each other.
13. Primary antibody was drained off the section and slides were flooded for 2 times
in 1 X PBS for 5 minutes each.
14. Add Secondary antibody conjugate onto the sections and keep for 15 minutes
at room temperature .
17. Secondary antibody was drained off and slides were washed with 1XPBS twice for 5 minutes each.
18. 100 ul of DAB mixture (1 ml of DAB substrate and 20 ul of DAB chromogen is added ) and incubated in dark for 5 min. Then the sections are given two washes with 1XPBS for 5 min each.
19. Dipped in Hematoxalin for a minute
20. Rinsed with Dist. Water
21. Kept under Tap water for 5 minutes(to allow stain to develop)
21. Counterstaining:Slides were dipped in 90% ethanol 3 times for a period of
5 minutes. Followed by 100% ethanol 3 times for 5 minutes. Slides were then
dipped in Xylene 2 times for a period of 5 minutes each.
22. Mounting:A drop of Permount was placed on the slide using a glass rod. The
coverslip was gently placed on to the slide to allow the Permount to spread
beneath the coverslip, covering the whole tissue.
23. Slides were dried overnight in the hood.
Analysis:
Slides were then analyzed for AIF expression. Semi quantitative scoring was performed
at a magnification of X400. From each section, five regions were captured using charge coupled device camera attached to Nikon E400 microscope (Japan). Expression was scored on the basis of intensity as follows: 0, absent or no expression 0.5, mild expression 1, moderate expression 2, high expression and 3, intense expression. Two independent, unbiased scorers who were blinded to the origin of the histological specimen performed the analysis. Different sections from each tumor and normal sample were scored, and a mean of that was taken for analysis. Any discrepancies in scores were resolved by discussion with a pathologist.
NORMAL TUMOR
AIF : Abcam 1998 polyclonal antibody
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
MRS. Thushara Jayan Pillai
Verified customer
投稿 Oct 24 2011