Paraffin Sections
Sacrifice the animal and remove the tissue of interest.
Fix in 10% formalin for no more than 24 hours at 4°C.
Transfer to 70% ethanol for long-term storage.
Submit sample for paraffin embedding.
Cut 4-6 µm sections on a microtome and dry slides as usual.
Deparaffinize using the automated slide processor at the core.
Do not allow slides to dry out afterward—keep them in PBS or tap water.
Perform antigen retrieval:
Immerse slides in Citrate Buffer (pH 6.0).
Place slides in a pressure cooker with water in the bottom.
Run the standard program.
When finished, remove slides and cool them by immersing in water.
Immunofluorescence Staining
Using a PAP pen, draw an unbroken hydrophobic barrier around the tissue. Allow a few minutes to dry completely. Do not touch the tissue with the pen, and do not let the tissue dry out.
Wash slides 3 × 5 minutes in Wash Buffer at room temperature.
Add Blocking Buffer and incubate 60–120 minutes at room temperature.
Wash slides 3 × 5 minutes in Wash Buffer.
Add freshly prepared Permeabilization Buffer and incubate for 30 minutes. Do not exceed this duration. Permeabilization may not be needed for thin sections.
Wash slides 3 × 5 minutes with Wash Buffer.
Prepare Primary Antibody Dilutions and apply 30–60 µL per section.
Incubate in a dark humidified chamber (60–120 minutes at room temperature, or 12–16 hours at 4°C).
Wash 3 × 5 minutes with Wash Buffer to remove unbound antibody.
Prepare Secondary Antibody Dilutions and apply to slides.
Incubate in a dark humidified chamber (60–120 minutes at room temperature, or 12–16 hours at 4°C)
Wash slides 3 × 3 minutes in Wash Buffer.
Add Tissue Autofluorescence Reagent and incubate 5 minutes at room temperature.
Wash slides 3 × 3 minutes in Wash Buffer.
Incubate with DAPI for 5 minutes in the dark at room temperature.
Wash slides 3 × 3 minutes in Wash Buffer.
Pour off excess buffer and briefly rinse with PBS.
Wipe off excess PBS carefully without disturbing tissue.
Mount with Mounting Medium:
Add a drop directly onto the tissue.
Gently place coverslip using a razor blade trick to avoid bubbles.
A syringe can help dispense medium without bubbles.
Allow slides to dry at room temperature until mounting medium sets.
Store finished slides at 4°C for imaging.
Solutions and Reagents
0.5% v/v Tween 20 in PBS (Example: 5 mL Tween-20 in 995 mL PBS)
Blocking Buffer
2% w/v BSA and 10% v/v Normal Goat Serum (NGS) in PBS (Example: 0.4 g BSA + 2 mL NGS + PBS to 20 mL)
Antibody Diluent (Goat Host)
2% v/v NGS in Wash Buffer (Example: 0.4 mL NGS + 19.6 mL Wash Buffer)
Antibody Diluent (Other Hosts)
1% w/v BSA in Wash Buffer (Example: 0.2 g BSA + PBS to 20 mL)
Permeabilization Buffer (Prepare Fresh)
2% v/v Triton X-100 in PBS (Example: 0.2 mL Triton X + 9.8 mL PBS)
Tissue Autofluorescence Reagent
Mix equal volumes of A + B, then add an equal volume of C. Use within 1 hour.