Histology Stains

Hematoxylin & Eosin

• Bring sections to distilled water.
• Stain nuclei with the alum haematoxylin for 5 minutes.
• Rinse in running tap water.
• Differentiate with 0.3% acid alcohol for 1 minute.
• Rinse in running tap water.
• Rinse in Scott's tap water substitute for 1 minute.
• Rinse in tap water.
• Stain with eosin for 2 minutes.
• Dehydrate, clear and mount.

Results

• Collagen = pale pink
• Muscle = deep pink
• Acidophilic cytoplasm = red
• Basophilic cytoplasm = purple
• Nuclei = blue
• Erythrocytes = cherry red

Periodic Acid Schiff's (PAS)

• Bring sections to water.
• Oxidise with 1% periodic acid 5 mins (* 10 mins.)
• Wash in water 1 min.
• Place in Schiff's reagent at room temp. 10 min (* 20 mins.)
• Transfer to sulphite rinse (Optional).
• Wash well in running tap water 10 mins.
• Counterstain in Mayer's haematoxylin 30-45 secs.
• Rinse in tap water.
• Blue in Scott's tap water.
• Wash in water.
• Dehydrate, clear and mount in DPX.

Results

• PAS positive material = magenta/pink
• Nuclei = blue

Note

• Times in brackets are for refrigerated solutions.

Masson's trichrome

• Bring sections to water.
• Mordant sections in Bouin's fluid for 1 hour at 60° C (or overnight at room temperature)...Rinse in tap water, without removing all the yellow from the section.
• Stain with Celestine Blue 5 mins.
• Wash in water.
• Stain in Mayer's Haematoxylin 5 mins.
• Wash in water.
• Differentiate nuclei with Acid Alcohol, leaving the nuclei fairly overstained as the...subsequent stains will also remove some nuclear staining as they contain acid.
• Wash in water.
• Blue in Scott's.
• Wash in water.
• Stain in Biebrich Scarlet/Acid Fuchsin 15 mins.
• Wash in water.
• Treat with 5% Phosphotungstic Acid 20 mins.
• Wash in water.
• Stain in Aniline blue 20 mins.
• Drain off Aniline blue. DO NOT WASH OFF WITH WATER...Cover with 1% Acetic Acid 3 mins.
• Dehydrate, clear and mount in DPX.

Results

• Collagen fibers = blue
• Cytoplasm and Muscle = red
• Nuclei = dark blue to black

Picrosirius Red

• De-wax and hydrate paraffin sections.
• Stain in picro-sirius red (Solution A) for 1 hour
• Wash in two changes of acidified water (Solution B)
• Physically remove most of the water from the slides by vigorous shaking or (for a few slides only) blotting with damp filter paper.
• Dehydrate in three changes of 100% ethanol
• Clear in xylene and mount in a resinous medium

Results

• In bright-field microscopy collagen is red on a pale yellow background.

Methenamine Silver Staining

• Deparaffinize slides to distilled water.
• Oxidize in 0.5% periodic acid solution for 15 minutes at room temperature.
• Rinse 3 times in distilled water.
• Incubate slides in methenamine silver working solution for 30 minutes-1 hour at 60 degrees C.
• Rinse in hot distilled water and check microscopically.
• Rinse in distilled water at room temperature.
• Tone in gold chloride solution for 1 minute.
• Rinse in distilled water.
• Treat with sodium thiosulfate solution for 2 minutes.
• Wash in running tap water for 10 minutes.
• Counterstain in nuclear fast red or light green for 5 min.
• Dehydrate, clear in xylene and coverslip using a synthetic mounting medium.

Results

• Basement membranes, reticular fibers = black
• Nuclear or background = pink or green

Oil Red O

• Cut frozen sections at 8 to10mm, air dry the sections to the slides
• Fix in formalin, briefly wash with running tap water 1-10 mins
• Rinse with 60% isopropanol
• Stain with freshly prepared Oil Red O working solution 15 mins
• Rinse with 60% isopropanol
• Lightly stain nuclei with alum haematoxylin 5 dips
• Rinse with distilled water
• Mount in aqueous mountant or glycerine jelly.

Results

• lipid = red
• nuclei = blue