Author Topic: APAAP Method (VectorBlue) for Paraffin Sections  (Read 3524 times)

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Offline ihcwor2

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APAAP Method (VectorBlue) for Paraffin Sections
« on: March 12, 2003, 08:01:05 PM »
APAAP Method (VectorBlue) for Paraffin Sections


Solutions and Reagents

A. TBS Buffer (10mM Tris-HCl, 150mM NaCl, pH 7.5):
Tris-HCl (MW 157.60) --------------------- 1.58 g
NaCl (MW 58.44) ---------------------------- 8.77 g
Distilled water -------------------------------- 1000 ml
Mix to dissolve and adjust pH to 7.5

B. AP Buffer (100mM Tris-HCl, 0.9% NaCl, pH 8.2):
Tris-HCl (MW 157.60) --------------------- 15.8 g
NaCl (MW 58.44) ---------------------------- 9.0 g
Distilled water -------------------------------- 1000 ml
Mix to dissolve and adjust pH to 8.2

C.   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.

D.   Levamisole Solution:

E.   Blocking Solution:
2% Normal Serum and 1% BSA in TBS:
To prepare 100 ml
Normal serum ---------- 2 ml
BSA ----------------------- 1 g
TBS ----------------------- 100 ml
Stir to dissolve.

F.   Primary Antibody:
Mouse or rabbit primary antibody.

G.   Secondary Antibody:
Unconjugated anti-mouse or rabbit secondary antibody.

H.   APAAP Reagent:

I.   VectorBlue 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: (Select an appropriate antigen retrieval technique and it depends on antibodies used).
6.   Rinse sections in TBS for 1x5min.

7.   Blocking: incubate sections with blocking solution for 30 minutes to block non-specific binding of secondary immunoglobulin.
8.   Rinse in FBS for 1x2min.                                                                        

9.   Primary antibody: incubate sections with mouse or rabbit primary antibody diluted in blocking solution for 1 hour at room temperature (A titer must be performed prior to actual projects)
10.   Rinse in TBS 3x5 min.

11.   Secondary antibody: incubate sections with unconjugated anti-mouse or rabbit secondary antibody in TBS for 30 minutes at room temperature.
12.   Rinse in TBS for 3x5min.

13.   APAAP: incubate sections with mouse or rabbit APAAP complex reagent in TBS for 30 minutes at room temperature.
14.   Rinse in TBS for 3x5min.

15.   VectorBlue: incubate sections with VectorBlue solution containing 1mM Levamisole for 20-30 minutes
16.   Rinse in distilled water 2x5min.

17.   Counterstain with nuclear fast red if desire.
18.   Rinse in distilled water 2x5min.

21.   Coverslip with aqueous mounting medium.


Results:

1.   Positive staining ------------------------ blue
2.   Nuclei -------------------------------------- red

APAAP Method (VectorBlue) for Paraffin Sections
« on: March 12, 2003, 08:01:05 PM »