ImmunostainingofParaffinSections

Procedure:

1) 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

2)  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.

3) 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]. 

4) 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.

5) 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.

6) 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, then 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.

7)  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.