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P r o t o c o l s

Immunohistochemistry / Tissue Section Staining

Preparing Tissue Sections for Immunostaining

  1. Fix the tissue in 10% formalin at 4°C overnight.
  2. Paraffin embed the fixed tissue.
  3. Mount tissue sections on slides.
  4. Clear the paraffin with xylene for ten minutes; move slides to a fresh dish of xylene for an additional ten minutes.
    NOTE: Perform all xylene washes in a fume hood!
  5. Rinse the slides twice for 2 minutes in 100% alcohols (18:1:1 100% ethanol:100% methanol:100% isopropanol).
  6. Rinse the slides twice for 2 minutes in a 95% solution of the 100% alcohols.
  7. Place slides in an 80% solution of the 100% alcohols for 2 minutes, followed by deionized water for 5 minutes.
  8. Rinse slides several times with fresh deionized water followed by another five minute wash using fresh water.

SDS Antigen Retrieval Method

  1. Place slides face-up in incubation tray and cover each section with 1% SDS in TBS (100mM Tris pH 7.4, 138mM NaCl, 27mM KCl).
  2. Incubate for five minutes at room temperature, followed by three five minute washes with TBS.

Blocking

  1. Immerse slides in a dish containing blocking buffer (serum from host species of secondary antibody to be used, diluted 1:10 in TBS).
  2. Incubate at 37°C for one hour.

Incubation with Primary Antibodies

  1. Cover the tissue sections with primary antibody diluted in blocking buffer. An initial antibody concentration of 1.0–10 µg/ml is recommended.
  2. Incubate for 2 hours at 37°C.
  3. Blot excess liquid from slides and rinse three times in TBS for five minutes each wash.

Incubation with Secondary Antibodies

  1. Cover the tissue sections with secondary antibody diluted in blocking buffer according to manufacturer’s instructions. We routinely use an affinity purified donkey anti-mouse IgG conjugated to Cy5 (Jackson ImmunoResearch Laboratories).
  2. Incubate at 37°C for one hour.
  3. Blot excess liquid and rinse twice in TBS for five minutes each wash.

Counterstaining and Visualization

Nuclear Staining

  1. Immerse slides for 1–2 minutes in a solution of ethidium bromide diluted to 0.5–1.0 µg/ml in TBS.
  2. Rinse several times in deionized water. Blot excess water around tissue, then apply one drop of mounting media to tissue and place coverslip over slide. Seal with nail polish.
  3. The primary antibody signal is detected at 680nm and the nuclear staining at 585nm.

Alternate Method

  1. The Vecta Stain Elite kit from Vector Laboratories can also be used for detection of the primary antibody signal. Follow the guidelines recommended by the manufacturer. Cover the tissue sections with the DAB substrate/chromagen solution for approximately 1–8 minutes and visualize any color change.
  2. Wash in deionized water. Counterstain if desired and mount for microscopy.

Reference

Brown, D., et al. 1996 Histochem Cell Biol 105:261–267

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