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Author Topic: Indirect Immuno-POD Method (NovaRed) for Frozen Sections  (Read 1989 times)

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Offline ihcwor2

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Indirect Immuno-POD Method (NovaRed) for Frozen Sections
« on: March 16, 2003, 06:04:31 PM »
Indirect Immuno-POD Method (NovaRed) 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.   3% Hydrogen Peroxide:
To prepare 100 ml,
30% H2O2 ---------------- 10 ml
PBS or methanol ------- 90 ml

C.   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.

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:
Peroxidase conjugated secondary antibody

F.   NovaRED Reagent

       
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.   Hydrogen Peroxide: incubate sections in 3% H2O2 in PBS (or methanol) for 10-15 minutes to block endogenous peroxidase activity.
6.   Rinse in PBS for 1x2min.

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

9.   Primary antibody: incubate sections with primary antibody diluted in blocking solution 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 peroxidase conjugated secondary antibody in PBS for 30 minutes at room temperature.
12.   Rinse in PBS for 3x5min.

13.   NovaRED: incubate sections with NovaRED solution for 2-10 minutes.
14.   Rinse in distilled water 2x5min.

15.   Counterstain with hematoxylin if desire.
16.   Rinse in distilled water 2x5min.
17.   Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
18.   Clear in xylene for 2x5min.

19.   Coverslip with mounting medium.

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

Indirect Immuno-POD Method (NovaRed) for Frozen Sections
« on: March 16, 2003, 06:04:31 PM »