ICC Using Vectastain Kit

 

Day 1 ~ at RT except where indicated

 

1.       Deparaffinize in xylene 2X 3 min each OR etch in saturated EtOH/NaOH for 15 min (for plastic sections only).

2.      Rehydrate sections in graduated alcohol concentrations:

100% ~ 2X 3 min each

95% ~ 2X 3 min each

70% ~ 1X 3 min each

3.      Following rehydration, batch rinse in TBS 3X 3 min each in staining dishes.

4.      Dry slides and apply PAP Pen circles around sections.

5.      Apply 30% H2O2 for 5 min dropwise on sections to quench endogenous peroxidase activity.

6.      Following peroxide, batch rinse in TBS 3X 3 min each.  Dry slides.

7.      Incubate in blocking solution (3 drops normal serum/10 ml TBS) for 30 min dropwise on slides.

8.      Following blocking, batch rinse slides 3X 3 min each.  Dry slides.

9.      Incubate dropwise in primary antiserum diluted in 1% normal serum (2 drops of serum/10 ml TBS) overnight at 4° C in a humid chamber.  Always put preimmune control serum on sections closest to frosted end of the slide and the immune serum on sections at the other end of the slide.

 

Day 2 ~ at RT

 

1.       Following first antibody, batch rinse in TBS 3X 3 min each.  Dry slides.

2.      Incubate dropwise in second (biotinylated) antibody for 30 min.  (1 drop biotinylated antibody/10 ml TBS).

3.      As soon as slides are in second antibody, prepare ABC reagent and let sit for 30 min. before using.  (2 drops reagent A/10 ml TBS.  Mix well.  Add 2 drops reagent B and mix well).

4.      Following second antibody, batch rinse in TBS 3X 3 min each.  Dry slides.

5.      Incubate dropwise in ABC reagent for 60 min.

6.      Following ABC reagent, Batch rinse in TBS 3X 3 min each.

7.      Prepare DAB solution.  CAUTION: DAB IS A CARCINGEN.  HANDLE WITH GLOVES! 

      DAB is made up in 30 mg/ml TBS aliquots and frozen.

4 ml stock DAB

196 ml TBS Vacuum filter with Whatman #1 using Buchner funnel and Erlenmeyer flask in the hood.  Just prior to use, add 25 ul 30% H2O2.

8.      Incubate in DAB in a staining dish for 5 min for paraffin (30 min for plastic).Note:  Time in DAB is relative to the antibodies and concentrations being used.  For some experiments, longer or shorter times may be necessary.  To check development, let sit in DAB for 5 min, rinse briefly in TBS and check color development under the microscope.  If developed enough, go on to next step.  If underdeveloped, put back in DAB.

9.      Following DAB, batch rinse in TBS 2X 3 min each; dH2O 1X 3 min.

10.   Dehydrate sections originally embedded in paraffin in the following series of alcohols:

70% ~ 1X 3 min

95% ~ 2X 3 min each

100% ~ 2X 3 min each

11.    Place in xylene 2X 3 min each and individually mount coverslips with permount.  For plastic sections, skip dehydration and xylene and mount coverslip with permount.

 

 

Solutions

 

1.       20X TBS stock.  Use to prepare 1X TBS used in ICC.

           50 mM Tris, 0.15 M NaCl, pH 7.6

            121 g Tris base

            175 g NaCl

            qs to 1 L dH2O

            pH to 7.6 and store at 4 C.

 

2.      Saturated EtOH/NaOH

300 ml 100% EtOH

Add NaOH pellets and stir to dissolve about 1 hr.  If all NaOH dissolves, add more until the solution is saturated.  Allow solution to settle before using.  Can be made the night before.  Do not allow sediment to pour into staining dish.

 

3. DAB Stock (30 mg/ml)

            1 5 g bottle of 3,3'-diaminobenzidine (Sigma D-5637)

            166.7 ml 1X TBS

            Aliquot into 3 and 4 ml tubes.  Store at -20 C.