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Author Topic: ABC-POD Method (AEC) for Frozen Sections  (Read 2262 times)

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Offline ihcwor2

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ABC-POD Method (AEC) for Frozen Sections
« on: March 16, 2003, 05:44:52 PM »
ABC-POD Method (AEC) 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:
An appropriate biotinylated secondary antibody should be selected and a titration is needed prior to application of actual projects.

F.   ABC Reagent:
Depending on sensitivity and morphological requirements, there are varieties of ABC kits and reagents can be selected from. The most commonly used one is VECTASTAIN ABC Kit from Vector Laboratories.      

G.   AEC 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 biotinylated secondary antibody in PBS for 30 minutes at room temperature.
12.   Rinse in PBS for 3x5min.

13.   ABC: incubate sections with ABC complex or HRP-streptavidin reagent in PBS for 30 minutes at room temperature.
14.   Rinse in PBS for 3x5min.

15.   AEC: incubate sections with AEC solution for 2-10 minutes.
16.   Rinse in distilled water 2x5min.

17.   Counterstain with hematoxylin if desire.
18.   Rinse in distilled water 2x5min.

19.   Coverslip with aqueous mounting medium.


Results

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

ABC-POD Method (AEC) for Frozen Sections
« on: March 16, 2003, 05:44:52 PM »