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Author Topic: Immunofluorescence Double Staining P2 for Frozen Sections  (Read 1729 times)

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Offline ihcwor2

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Immunofluorescence Double Staining P2 for Frozen Sections
« on: March 16, 2003, 06:17:59 PM »
Immunofluorescence Double Staining P2 (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 from mouse and the other is from rabbit.

D.   Secondary Antibody:
      FITC conjugated horse anti-mouse secondary antibody.
            Texas Red conjugated goat anti-rabbit secondary 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.   Secondary antibody: incubate sections with the mixture of the two secondary antibodies in PBS for 30 minutes at room temperature.
10.   Rinse in PBS for 3x5min.

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

Immunofluorescence Double Staining P2 for Frozen Sections
« on: March 16, 2003, 06:17:59 PM »