Immunostaining of Paraffin Sections¶
Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
Immunostaining of Paraffin Sections
- Fix tissues for 3 hr on ice in 4% formaldehyde (2.5 ml of Polysciences #18814 made up to 10 ml in 80 mM NaPO4 [3.2 ml of 1 M NaPO4] pH 6.8 containing 0.2 gm of sucrose).
- Wash for 2 hr in several changes of cool PBS.
- Infiltrate and embed in paraffin (use automatic processor). Embed in flat end of capped Beem capsules
- Prepare polylysine-coated slides by immersing clean slides, via a stainless steel slide rack, in 400 ml of 50 µg/ml polylysine (Sigma #P1524; make up in 10 mM Tris, pH 8) for 30 min.
- Cover with foil and let dry overnight at room temperature. Polylysine solution can be stored at -20°C and reused. Store coated slides at 4°C and use within 4 wks.
- Trim away excess paraffin so that block face is 2 - 3 mm2.
- Set up on an ultramicrotome using glass knives or an old diamond knife with ‘boat’ filled with water. Rapidly cut away surface paraffin, then cut 1 - 2 µm sections.
*Sections cut easily and should form ribbons. *
- Pickup with the point of a 25 gauge needle and transfer to a drop of water on a polylysine-coated slide. Have two groups of sections per slide. Transfer slide to a 42°C warming plate and leave overnight.
Sections prepared in this manner can also be used for in situ hybridization
- Deparaffinize sections in xylene (2 x 10 min) using glass slide jars. Transfer to 100% EtOH (2 x 5 min), and then to PBS (2 x 5 min).
- Insert sections in Coplin jars containing ‘TUF’ (Target Unmasking Fluid; Signet Labs [Dedham MA] #1050) preheated to 80-90°C.
- Incubate in TUF for 10 min at 80-90°C.
- Let jar cool 5 - 10 min, then rinse two times in ddH2O and two times in PBS.
- Place sections on a staining rack and block with PBS containing 1% BSA for 60 min at room temperature.
- Pour off, dry with kimwipe around sections. Add diluted (dilute in PBS containing 1% BSA; use 10 fold dilutions at start) antibody or preimmune control in one or two drops to sections
- Place slides in a closed 150 mm petri dish containing wet filter paper, and place overnight at 4°C.
- The next day, wash five times over 40 min in PBS containing 1% BSA.
- At this time, can immerse in Pierce ‘peroxidase suppressor’ (Pierce #35000) for 30 min at room temperature.
- Wash several times in PBS containing 1% BSA using squirt bottle to carefully wash around sections. Dry around sections, then add one or two drops of secondary peroxidase-labeled antibody diluted 1/1,000 in PBS containing 1% BSA.
- Incubate for 60 min at room temp on staining rack.
- Wash five times over 40 min in PBS using squirt bottle.
- Dry around sections, then add Pierce DAB metal concentrate (Pierce #1856090) diluted 1/10 in stable peroxide substrate buffer (Pierce #1855910).
- Allow reaction to go for 5 - 15 min, then wash with ddH2O using squirt bottle.
- Dehydrate slides in 80%, 90% and 2x 100% EtOH (1 min each), then immerse in 2 changes of xylene (1 min each).
- Mount cover slips on the slides using Permount (Fisher) and examine in the light microscope.
This method is based, with permission, on an original protocol available here.