Abcam Ad

Author Topic: Indirect Immuno-AP Method (VectorBlue) for Frozen Sections  (Read 2001 times)

0 Members and 1 Guest are viewing this topic.

Offline ihcwor2

  • Administrator
  • GoldMember
  • *****
  • Posts: 335
    • http://www.ihcworld.com/
Indirect Immuno-AP Method (VectorBlue) for Frozen Sections
« on: March 16, 2003, 06:00:18 PM »
Indirect Immuno-AP 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.

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:
Alkaline phosphatase conjugated secondary antibody

F.   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 alkaline phosphatase conjugated secondary antibody in TBS for 30 minutes at room temperature.
10.   Rinse in TBS for 3x5min.

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

13.   Counterstain with nuclear fast red if desire.
14.   Rinse in distilled water 2x5min.

15.   Coverslip with aqueous mounting medium.

Results

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

Indirect Immuno-AP Method (VectorBlue) for Frozen Sections
« on: March 16, 2003, 06:00:18 PM »