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Author Topic: ABC-AP Method (VectorBlack) for Frozen Sections  (Read 2748 times)

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Offline ihcwor2

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ABC-AP Method (VectorBlack) for Frozen Sections
« on: March 16, 2003, 05:40:39 PM »
ABC-Alkaline Phosphatase Method (VectorBlack) 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-Cl, 100mM NaCl, 5mM MgCl2, pH 9.5)
Tris-HCl (MW 157.60) --------------------- 15.76 g
NaCl (MW 58.44) ---------------------------- 5.84 g
MgCl2 (MW 203.31 -------------------------- 1.02 g
Distilled water --------------------------------- 900 ml
Mix to dissolve. Adjust pH to 9.5 and then bring total volume to 1 liter.

C.   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.

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-AP Kit from Vector Laboratories.      

G.   VectorBlack Reagent


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.   VectorBlack: incubate sections with VectorBlack solution containing 1 mM Levamisole for 2-10 minutes
14.   Rinse in distilled water 2x5min.

15.   Counterstain with nuclear fast red 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.


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

ABC-AP Method (VectorBlack) for Frozen Sections
« on: March 16, 2003, 05:40:39 PM »