Download a listing of our antibodies and protocols
CAT-I Basic Staining Protocol
- Remove slides from -20°C. Dry tissue at RT for at least 1 hour in a humidity chamber.
- Outline tissue with an ImmEdge Hydrophobic Barrier PAP Pen (Vector Labs H-4000)
- Rehydrate tissue with 1X PBS (sterile). Let sit for 10 minutes at RT.
- Block tissue with blocking/staining buffer (1% BSA, 0.3% Triton-X-100, and 1% mouse or human sera). Block for at least 1 hour at RT. Note: Filter blocking buffer with 0.22 mm filter and keep sterile.
- Dilute primary antibodies in blocking buffer. Stain overnight at 4°C.
- Wash tissue 3X with sterile PBS, 5-10 minutes per wash.
- Dilute secondary antibodies in blocking buffer. Stain for 4 hours at RT or overnight at 4°C.
- Wash tissue 3X with sterile PBS, 5-10 minutes per wash.
- Apply mounting media to samples before placing coverslip on tissue
- Store at RT in dark for several weeks
Fixation Protocol
We are working to optimize fixation protocols for various tissues. Depending on the tissue, we have emerging protocols using BD Cytofix/Cytoperm, PFA, FFPE, and snap frozen prepared tissues. Please contact CAT-I staff for additional details.
Materials
- BD Cytofix solution (BD Cat #554722)
- Sucrose (Sigma Cat #S2903)
- OCT (Tissue Tek Cat #4583)
- Superfrost glass slides (VWR Cat #48311-703)
Protocol
- Carefully remove all fat and connective tissue from sample. Fix sample in BD Cytofix solution (1 to 4 dilution in 1X PBS).
- Completely submerge tissue in fixative and incubate at 4ºC for 1-5 days.
- After fixation, wash tissue 2 times in PBS for 5-15 minutes.
- For Ce3D-cleared tissue: Stop at Step 3
- For confocal imaging and Histo-cytometry: Continue to Steps 4-6
- Incubate fixed tissues in 30% sucrose (prepared in PBS) at 4ºC for 1-5 days or until the tissues sink. Note: Prepare sucrose in sterile PBS, filter, and keep sterile.
- Embed tissue in OCT, store blocks at -80ºC.
- Prepare 20-30 µm sections with a cryostat.
Please contact CAT-I staff for additional details.