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Author Topic: NOS2 Immunoenzyme Staining Protocol for Paraffin Sections  (Read 1601 times)

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Offline ihcwor2

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NOS2 Immunoenzyme Staining Protocol for Paraffin Sections
« on: April 17, 2003, 11:35:46 AM »
NOS2 Immunoenzyme Staining Protocol for Paraffin Sections


Solutions and Reagents

A. 10X Phosphate Buffered Saline (10xPBS):
To prepare 1 liter,
Na2HPO4 ------------------ 10.9 g
NaH2PO4 ------------------ 3.2 g
NaCl ------------------------ 90 g
Distilled water ----------- 1000 ml
Mix to dissolve and adjust pH to 7.4

B. 3% Hydrogen Peroxide:
To prepare 100 ml,
30% H2O2 ---------------- 10 ml
PBS or methanol ------- 90 ml

C. Blocking Solution:
2% Normal Goat Serum in PBS:
To prepare 100 ml
Normal goat serum ------ 2 ml
PBS --------------------------- 98 ml
Mix to dissolve.

D. Primary Antibody:
Rabbit anti-NOS2 (Santa Cruz, Cat# sc-651). Optimal dilution 1:400.

E. Secondary Antibody:
Goat anti-rabbit IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-1000). Optimal dilution 1:400.

F. ABC Reagent:
HRP-streptavidin (Vector Laboratories, Cat# HA-5004). Optimal dilution 1:400.

G. DAB Reagent:
0.02% DAB and 0.003% H2O2 in PBS
To prepare 100 ml
DAB ----------------------- 20 mg
PBS ----------------------- 100 ml
Stir to dissolve. Add 10 ul of 30% H2O2 and filter. Use the solution immediately.


Procedure

1. Deparaffinize sections in xylene for 3x5min.
2. Hydrate with 100% ethanol for 2x5min.
3. Hydrate with 95% ethanol for 2x5min.
4. Rinse in distilled water.

5. Perform Microwave-citrate buffer antigen retrieval.

6. Hydrogen Peroxide: incubate sections in 3% H2O2 in PBS (or methanol) for 10-15 minutes to block endogenous peroxidase activity.
7. Rinse in PBS for 1x2min.

8. Blocking: incubate sections with 2% normal goat serum in PBS for 20 minutes to block non-specific binding of secondary immunoglobulin.

10. Primary antibody: incubate sections with rabbit anti-NOS2 diluted 1:400 in PBS for 1 hour or 1:3000 overnight at 4 C.
11. Rinse in PBS 3x5 min.

12. Secondary antibody: incubate sections with biotinylated goat anti-rabbit IgG diluted 1:400 in PBS for 30 minutes at room temperature.
13. Rinse in PBS for 3x5min.

14. ABC: incubate sections with HRP-streptavidin reagent diluted 1:400 in PBS for 30 minutes at room temperature.
15. Rinse in PBS for 3x5min.

16. DAB: incubate sections with DAB solution for 2-10 minutes.
17. Rinse in distilled water 2x5min.

18. Counterstain with hematoxylin if desire.
19. Rinse in distilled water 2x5min.
20. Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
21. Clear in xylene for 2x5min.

22. Coverslip with mounting medium.


Results

1. NOS2 positive sites -------------------------- brown
2. Nuclei ----------------------------------------- blue

NOS2 Immunoenzyme Staining Protocol for Paraffin Sections
« on: April 17, 2003, 11:35:46 AM »