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Author Topic: Immunofluorescence Double Staining P1 for Paraffin Sections  (Read 2551 times)

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Offline ihcwor2

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Immunofluorescence Double Staining P1 for Paraffin Sections
« on: March 12, 2003, 09:14:44 PM »
Immunofluorescence Double Staining P1 (FITC&TexasRed) for Paraffin Sections


Solutions and Reagents

A.   10X Phosphate Buffered Saline (PBS):
To prepare 1000 ml, add 10.9 g Na2HPO4, 3.2 g NaH2PO4 and 9 g NaCl to 1000 ml distilled water. 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, add 2.94 g sodium citrate to 1000 ml distilled water. 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 before application to actual projects.

C.   Blocking Solution:
2% normal serum and 1% BSA in PBS:
To prepare, add 2 ml of normal serum and 1 g BSA to 100 ml PBS. Stir to dissolve.

D.   Primary Antibody:
Two primary antibodies must be raised from different species. For example, one is FITC conjugated mouse antibody and the other is Texas Red conjugated rabbit antibody.

       
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 PBS for 1x5min.

7.   Blocking: incubate sections with 2% normal serum in PBS (Use serum mixture if secondary antibody are from different species).
8.   Rinse in PBS for 1x2min.

9.   Primary antibody: incubate sections with the mixture of the two primary antibodies diluted in PBS for 1 hour at room temperature or overnight at 4 C.
10.   Rinse in PBS 3x5 min.

11.   Counterstain if desired.
12.   Rinse 3x5min in PBS.

13.   Coverslip with aqueous mounting medium and seal with nail polish.

14.   Observation using a fluorescence microscope with appropriate filters. Store slides in the dark at 4 C.


Results

1.   One primary antibody staining ------------------ green
2.   The other primary antibody staining ----------- red

Immunofluorescence Double Staining P1 for Paraffin Sections
« on: March 12, 2003, 09:14:44 PM »