Author Topic: Immunoenzyme Double Staining Protocol for Paraffin Sections2  (Read 3200 times)

0 Members and 1 Guest are viewing this topic.

Offline ihcwor2

  • Administrator
  • GoldMember
  • *****
  • Posts: 335
    • http://www.ihcworld.com/
Immunoenzyme Double Staining Protocol for Paraffin Sections2
« on: March 12, 2003, 08:10:59 PM »
Immunoenzyme Double Staining Protocol for Paraffin Sections
(Method 2)


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.   Antigen Retrieval Solution:
10mM Sodium Citrate Buffer (The most commonly used antigen retrieval reagent):
To prepare 1000 ml,
Sodium citrate ---------------- 2.94 g
Distilled water ---------------- 1000 ml
Adjust pH to 6.0

Selection of antigen retrieval techniques is crucial for successful staining of paraffin sections. Different antibodies may require different antigen retrieval techniques and need to be tested prior to application of actual projects.

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

D.   Blocking Solution:
2% normal serum and 1% BSA in PBS:
To prepare 100 ml
Normal serum --------------- 2 ml
BSA ---------------------------- 1 g
PBS ---------------------------- 100 ml
Stir to dissolve.

E.   Primary Antibody:
Dilution of primary antibody is critical for success of staining, so a titration must be performed prior to application of actual projects.

F.   Secondary Antibody:
An appropriate biotinylated secondary antibody should be selected and a titration is needed prior to application of actual projects.
 
G.   ABC Reagent:
Depending on sensitivity and morphological requirements, there are varieties of ABC kits and reagents can be selected from. The most commonly used one is VECTASTAIN ABC Kit from Vector Laboratories.      

H.   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

I.   SG Reagent:

 
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.   Antigen Retrieval: (Depending on antibodies used).
6.   Rinse sections in PBS for 1x5min.

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

9.   Blocking: incubate sections with 2% normal serums in PBS.
10.   Rinse in PBS for 1x2min
                                                                                                                               
11.   1st Primary antibody: incubate sections with primary antibody diluted in PBS for 1 hour at room temperature (A titer must be performed prior to actual projects)
12.   Rinse in PBS 3x5 min.

13.   Secondary antibody: incubate sections with biotinylated secondary antibody in PBS for 30 minutes at room temperature.
14.   Rinse in PBS for 3x5min.

15.   ABC: incubate sections with ABC complex or HRP-streptavidin reagent in PBS for 30 minutes at room temperature.
16.   Rinse in PBS for 3x5min.

17.   DAB (brown): incubate sections with DAB solution for 2-10 minutes.
18.   Rinse in distilled water for 2x5min.
19.   Rinse in PBS for 1x5min.

20.   Repeat step 11 through 16. Apply the 2nd primary antibody and a matched biotinylated secondary antibody and incubate with the same ABC reagent.
21.   Rinse 3x5 min in PBS.

22.   Choose a different substrate (SG-blue) and incubate for 2-10 minutes.
23.   Rinse in distilled water for 2x5min.

24.   Counterstain with nuclear fast red if desire.
25.   Rinse in distilled water 2x5min.
26.   Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
27.   Clear in xylene for 2x5min.

28.   Coverslip with mounting medium.


Results:

1.   1st primary antibody staining ------------------ brown
2.   2nd primary antibody staining ------------------ blue
3.   Nuclei --------------------------------------------- pink

Immunoenzyme Double Staining Protocol for Paraffin Sections2
« on: March 12, 2003, 08:10:59 PM »