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Author Topic: PKA-alpha Immunoenzyme Protocol for Paraffin Sections  (Read 2160 times)

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Offline ihcwor2

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PKA-alpha Immunoenzyme Protocol for Paraffin Sections
« on: March 15, 2003, 02:25:18 AM »
PKA-alpha Immunoenzyme Staining Protocol for Paraffin Sections


Solutions and Reagents

A.   10X Phosphate Buffered Saline (PBS):
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-PKA-alpha (Santa Cruz Biotechnology Cat# sc-903). Optimal dilution 1:1000 in PBS.

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

7.   Blocking: incubate sections with 2% normal goat serum in PBS for 20 minutes to block non-specific binding of secondary immunoglobulin.
8.   Rinse in PBS for 1x2min.                                                                        

9.   Primary antibody: incubate sections with rabbit anti-PKA-alpha diluted 1:1000 in PBS for 1 hour at room temperature.
10.   Rinse in PBS 3x5 min.

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

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

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

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

21.   Coverslip with mounting medium.


Results

1.   PKA-alpha positive staining -------------- brown
2.   Nuclei ------------------------------------------- blue

PKA-alpha Immunoenzyme Protocol for Paraffin Sections
« on: March 15, 2003, 02:25:18 AM »