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Author Topic: Immunofluorescence Double Staining P1 for Frozen Sections  (Read 1708 times)

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Offline ihcwor2

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Immunofluorescence Double Staining P1 for Frozen Sections
« on: March 16, 2003, 06:15:57 PM »
Immunofluorescence Double Staining P1 (FITC&TexasRed) for Frozen 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.   Blocking Solution:
2% Normal Serum, 1% BSA and 0.2% Triton X-100 in PBS:
To prepare 100 ml
Normal serum ---------- 2 ml
BSA ----------------------- 1 g
Trition X-100 ------------ 0.2 ml
PBS ----------------------- 100 ml
Stir to dissolve.

C.   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.   Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at - 80 C.
2.   Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80 C until needed.

3.   Before staining, warm slides at room temperature for 5 minutes and fix in ice cold acetone for 10 minutes.
4.   Wash 3x2min in PBS.

5.   Blocking: incubate sections with blocking solution for 20 minutes.
6.   Rinse in PBS for 1x2min.                                                                        

7.   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.
8.   Rinse in PBS 3x5 min.

9.   Counterstain if desired.
10.   Rinse 3x5min in PBS.

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

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


Results

1.   primary antibody 1 staining -------------- green
2.   Primary antibody 2 staining -------------- red

Immunofluorescence Double Staining P1 for Frozen Sections
« on: March 16, 2003, 06:15:57 PM »