Core Facilities


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  • 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