Labeling Tissue Samples

Materials and Reagents

  • Blocking or Staining Buffer: (0.5% NP40, 10% DMSO, 0.5% Triton X-100,5% serum, 1X PBS)

  • Wash Buffer: (0.5%NP40, 10%DMSO, 1X PBS)

Procedure

Staining protocol

  1. Permeabilize and block tissue in blocking buffer with gentle shaking or rotation at room temperature overnight.

  2. Prepare primary antibody stock solution in staining buffer with the desire concentration one hour before tissue incubation.

  3. Incubate samples for 72 hours in primary antibody solution with gentle shaking or rotation at room temperature.

  4. Remove primary antibody with wash buffer 3x for 2 hours.

  5. Immerse samples in 4% PFA overnight at RT for primary post fixation.

  6. Wash out 4% PFA with 1X PBS twice for 10-15 minutes.

  7. Incubate samples for 72 hours in secondary antibody solution with gentle shaking or rotation at room temperature.

  8. Removed secondary antibody with wash buffer for at least two days changing the solution first day 3x every 2 hours and second day refreshed just one time.

  9. Nuclei staining. (Dilute dyes in 1XPBS) Incubate samples in nuclei dye for 48 hours at RT.

  10. Wash out nuclei dye with a few changes of PBS.

Notes and Cautions

  • We usually work with ~2mm tissue slices chopped with TED PELLA tissue slicer (0.5mm).

  • Serum should be from the species that secondary antibody is raised.

  • Important: Centrifuge the primary and/or secondary antibody for 2 seconds before aliquoting in staining buffer.

  • Homogenize antibody solution for 1 hour in a shaker at 4°C before staining.

  • 72 hours for ~2mm tissue size.

  • Protect samples from light with aluminum foil.