Tunel Staining in Paraffin Sections

 

Materials:

 

Apotag Plus Peroxidase In Situ Kit (Chemicon S7101)

 

*Please Note:  All tunnel assays are carried out in the reproductive core under the direction of Patty. 

 

Solutions:

1.  10% neutral buffered formalin

2.  PBS (50 mM sodium phosphate, pH 7.4, 200 mM NaCl)

3.  Hydrogen Peroxide, 30 % solution

4.  DAB in staining buffer (PBS or TBS)

5.  Proteinase K; prepare a 5 mg/ml stock in PBS and store in 150 µl frozen aliquots.

6.  0.5% methyl green, free of crystal violet

Optional: Triton X-100 10% stock solution, 10 mM citrate buffer, pH 6.0.

 

7.  Working Strength TdT Enzyme:  The concentrated TdT Enzyme is supplied in a stabilization buffer to preserve activity.  It must be diluted with Reaction Buffer prior to use.  Mix reagents in a ration of 70% Reaction Buffer to 30% TdT Enzyme. To prepare:  add in a fresh microfuge tube:  77 µl reaction buffer + 30 µl TdT Enzyme = 110 µl total.  Mix well by vortexing.  This reagent may be prepared in advance and stored on ice for no more than 6 hr.  This amount is enough to treat two 5 cm² tissue specimens.

 

8. Proteinase K volumes for Coplin Jar vs. direct slide application(d.s.a.):  Coplin Jar add 140 µl of stock to 35 ml of PBS; d.s.a. 60 µl of diluted stock per specimen.  Add 20 µl of stock to 5 ml PBS will provide enough volume to process more than 40 slides.

 

9.  Working Strength Hydrogen Peroxide:  Dilute to 3% in PBS.  For Coplin Jar, use 36 ml PBS and add 4 ml hydrogen peroxide.

 

10.  Working Strength Stop/Wash Buffer:  Add 1 ml (Stop/Wash Buffer) to 34 ml dH2O.  This amount (35 ml) is enough to treat 5 slides in a Coplin Jar.  This reagent may be prepared in advance and stored in a glass or plastic container at 4 C for up to 1 year.  Use a fresh aliquot for each experiment.

 

11.  Working Strength Peroxidase Substrate:  In clean tube add 147 µl DAB dilution buffer to 3 µl DAB substrate = 150 µl total.  Warm mixture at RT and store in dark until use.  This amount is enough to treat 2 tissue specimens.

 

12.  Methyl Green Counterstain for Nuclei:  0.5 % methyl green in 0.1 M sodium acetate, pH 4.0 (adjust with acetic acid). 

 

Procedure:

 

Do not allow sections to dry out during processing

 

  1. Deparaffinize Tissue Sections in a Coplin Jar under hood.
    1. Wash for 3 changes with xylene for 5 min each.
    2. Wash for 2 changes with 100% ethanol for 5 min each.
    3. Wash for 1 change with each: 95%, 70% for 3 min each.
    4. Wash for 1 change with PBS for 5 min.

 

 

  1. Pretreat Tissue
    1. Apply freshly diluted Protein Digestion Enzyme or Proteinase K (20 µl/ml) to the specimen for 15 min at RT in a Coplin Jar or directly on slide.
    2. Wash in 2 changes of dH2O in a Coplin jar for 2 min each.
  2. Quench Endogenous Peroxidases
    1. Quench in 3% hydrogen peroxide in PBS for 5 min. at RT either on slide or in a Coplin Jar.
    2. Rinse slides 2X with PBS or H2O 5 min each on a Coplin Jar.
  3. Apply Equilibration Buffer
    1. Gently tap off excess liquid and gently blot around the sections.
    2. Immediately apply 75 µl of equilibration buffer directly on the sections.
    3. Incubate for at least 10 sec at RT.  Slides may be left in equilibration buffer for up to 60 min at 4C or RT.
  4. Working Strength TdT Enzyme
    1. Gently tap off excess liquid and blot around the section.
    2. Immediately pipette onto section 55 µl/5 cm² of Working Strength TdT Enzyme.
    3. Incubate in a humid chamber at 37 C for 1 hr.
  5. Apply Stop/Wash Buffer
    1. Put the slides in a Coplin Jar containing Working Strength Stop/Wash Buffer.  Agitate for 15 sec then incubate for 10 min at RT.
    2. Remove an aliquot of Anti-Digoxigenin Conjugate from the stock vial sufficient to process the number of slides you have.  Warm aliquot to RT.
  6. Apply Anti-Digoxigenin Conjugate
    1. Wash the slides in 3X PBS for 1 min each.
    2. Gently tap off excess liquid and carefully blot around the sections.
    3. Apply RT Anti-Digoxigenin Conjugate to the slides.  Use about 65 µl/5 cm² of specimen surface area.
    4. Incubate in a humid chamber for 30 min at RT.
  7. Wash in PBS
    1. Wash the slides in 4X PBS in Coplin Jar for 2 min each at RT.
    2. While the slides are washing prepare Working Strength Peroxidase Substrate.
  8. Develop color in Peroxidase Substrate
    1. Gently tap off excess liquid and carefully blot around the sections.
    2. Apply enough Peroxidase Substrate to completely cover the section (75 µl/5cm²)
    3. Stain for 3-6 min. at RT.  A humidified chamber is not required.
    4. Monitor the color development by looking at the slides under the microscope.
  9. Wash Slides
    1. Wash slides in 3X dH2O in a Coplin Jar for 1 min each wash.  Longer washing will not destain the tissue.
    2. Incubate the slides in dH2O for 5 min at RT.
  10. Counterstain Specimens
    1. Counterstain in 0.5% methyl green in a Coplin Jar for 10 min at RT.
    2. Wash the slides in 3X dH2O in a Coplin Jar, dipping the slides 10X each in the first and second changes, followed by 30 sec without agitation in the third wash.
    3. Wash the slides in 3X 100% n-butanol in a Coplin Jar, dipping the slides 10 times each in the first and second changes, followed by 30 sec without agitation in the third wash.
  11. Mount the Specimens
    1. Dehydrate the tissue in 3X xylene for 2 min each.
    2. Remove slides one at a time, drain excess xylene and coverslip using regular mounting medium.

 

 

Slides may be stored indefinitely.