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Author Topic: Immunofluorescence Double Staining S2 for Paraffin Sections  (Read 2756 times)

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Offline ihcwor2

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Immunofluorescence Double Staining S2 for Paraffin Sections
« on: March 12, 2003, 09:23:56 PM »
Immunofluorescence Double Staining S2 (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 90 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:
Dilution of primary antibody is critical for success of staining, so a titration must be performed prior to application of actual projects.

E.   Secondary Antibody:
      Biotinylated secondary antibody.

F.   Streptavidin-FITC (green):
From Vector Labs and dilute 1:50 in PBS.

G.   Steptavidin-Texas Red:
From Vector Labs and dilute 1:50 in PBS.

       
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.   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)
10.   Rinse in PBS 3x5 min.

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

13.   Incubate sections in streptavidin-FITC (green) for 30 minutes, protecting the slide from light starting from here.
14.   Rinse 3x5min in PBS.
15.   Repeat step 9 through 12 using 2nd primary antibody and a biotinylated secondary antibody.

16.   Incubate sections in streptavidin-Texas Red for 30 minutes.
17.   Rinse 3x5 min in PBS.

18.   Counterstain if desired.
19.   Rinse 3x5min in PBS.

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

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


Results

1.   1st primary antibody staining ---------------- green
2.   2nd primary antibody staining ---------------- red
3.   Double staining --------------------------------- yellow

Immunofluorescence Double Staining S2 for Paraffin Sections
« on: March 12, 2003, 09:23:56 PM »