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Author Topic: ABC-AP Method (VectorBlue) for Frozen Sections  (Read 2473 times)

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Offline ihcwor2

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ABC-AP Method (VectorBlue) for Frozen Sections
« on: March 16, 2003, 05:42:02 PM »
ABC-Alkaline Phosphatase Method (VectorBlue) for Frozen Sections



Solutions and Reagents

A. TBS Buffer (10mM Tris-HCl, 150mM NaCl, pH 7.5):
Tris-HCl (MW 157.60) --------------------- 1.58 g
NaCl (MW 58.44) ---------------------------- 8.77 g
Distilled water -------------------------------- 1000 ml
Mix to dissolve and adjust pH to 7.5

B. AP Buffer (100mM Tris-HCl, 0.9% NaCl, pH 8.2):
Tris-HCl (MW 157.60) --------------------- 15.8 g
NaCl (MW 58.44) ---------------------------- 9.0 g
Distilled water -------------------------------- 1000 ml
Mix to dissolve and adjust pH to 8.2

C.   Levamisole Solution:

D.   Blocking Solution:
2% Normal Serum, 1% BSA and 0.2% Triton X-100 in TBS:
To prepare 100 ml
Normal serum ---------- 2 ml
BSA ----------------------- 1 g
Trition X-100 ------------ 0.2 ml
TBS ----------------------- 100 ml
Stir to dissolve.

E.   Primary Antibody:
Dilution of primary antibody is critical for success of staining, so a titration must be performed prior to application of actual projects.

F.   Secondary Antibody:
An appropriate biotinylated secondary antibody should be selected and a titration is needed prior to application of actual projects.

G.   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-AP Kit from Vector Laboratories.      

H.   VectorBlue 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 TBS.

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

7.   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).
8.   Rinse in TBS 3x5 min.

9.   Secondary antibody: incubate sections with biotinylated secondary antibody in TBS for 30 minutes at room temperature.
10.   Rinse in TBS for 3x5min.

11.   ABC: incubate sections with ABC-AP complex or AP-streptavidin reagent in TBS for 30 minutes at room temperature.
12.   Rinse in TBS for 3x5min.

13.   VectorBlue: incubate sections with VectorBlue solution containing 1 mM Levamisole for 20-30 minutes
14.   Rinse in distilled water 2x5min.

15.   Counterstain with nuclear fast red if desire.
16.   Rinse in distilled water 2x5min.

17.   Coverslip with aqueous mounting medium.


Results

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

ABC-AP Method (VectorBlue) for Frozen Sections
« on: March 16, 2003, 05:42:02 PM »