-
Hematoxylin-Eosin (H&E)
Toluol 3 x 5 min
Blot on absorbent paper
Isopropanol 3 x 5 min
tap water 5 min
distilled water 5 min
Blot on absorbent paper
Mayer Hematoxyline (VWR-Klinipath # VWRK64040/1) 10 min
tap water 5 min
distilled water 5 min
Blot on absorbent paper
Eosin Y solution 0.5 % in water (Carl Roth #X883.2) 4 min
tap water 30 sec
distilled water 30 sec
Air drying 15 min
Mounting: Sakura Tissue-Tek Film
► Result
nuclei: dark blue
cytoplasm: dark pink
collagen, elastin, erythrocytes: red-orange
-
Picro-Sirius red
Toluol 3x5 min
Blot on absorbent paper
Isopropanol 3 x 5 min
tap water 3 min
distilled water 3 min
Blot on absorbent paper
Phosphomolybdic acid (Roth #4440.3, keep powder at 4°C) 2% fresh 2min
tap water 3 min
distilled water 3 min
Blot on absorbent paper
Direct Red 80 (cfr below) 2 h protected from light (this solution can be re-used several times)
Blot on absorbent paper
HCl 0,01N fresh 2min
distilled water very quick bath (5 seconds)
Absolute methanol absolu very quick bath (3 x 10 up and down)
Blot on absorbent paper
Xylene 3 x 5 min
Mounting (Dako coverslipper)
► For frozen cuts :
Remove OCT in PBS 3x3 min
distilled water 3 min
Bouin 30 min
tap water 3 min
distilled water 3 min
Phosphomolybdic acid (Roth #4440.3) 1,5% fresh 2min
tap water 3 min
distilled water 3 min
Direct Red 80 (cfr below) 2 h protected from light
HCl 0,01N fresh 2min
distilled water very quick bath
Absolute methanol very quick bath
Blot on absorbent paper
Xylene 3 x 5 min
Mounting (Dako coverslipper)
► Picro-Sirius Red solution (100ml)
0,1 % Direct Red 80 (Sigma#365548) in a saturated picric acid solution (Sigma #P6744).
NB : prepare solution 24 hours before use so that the orange precipitate settles
► Result
collagen: red
background: yellow
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Masson's blue Trichrome
Toluol 3 x 5 min
Blot on absorbent paper
Isopropanol 3 x 5 min
tap water 3 min
distilled water 3 min
Blot on absorbent paper
Mayer Hematoxyline (Sigma # MHS32) 10 min
tap water 3 min
distilled water 3 min
Blot on absorbent paper
Ponceau-Fuschine (cfr below) 15 min
distilled water 1 quick bath (10 sec)
Blot on absorbent paper
Phosphomolybdic acid (Roth #4440.3) 1,5% fresh 5 min
distilled water 1 quick bath (10 sec)
Blot on absorbent paper
Bleu d’aniline (cfr below) 1 min
Blot on absorbent paper
Phosphomolybdic acid (Roth #4440.3) 1,5% fresh 5 min
distilled water 1 quick bath ( 10 sec )
Blot on absorbent paper
Methanol 3 quick bath ( 3 x 10 sec )
Blot on absorbent paper
Xylene 3x 5 min
Mounting (Dako coverslipper)
► Ponceau-Fuschine Solution :
1 g Ponceau – Xylidine (Sigma #P2395-25g) in 100 ml distilled water, bring to boil and after cooling add 1 ml glacial acetic acid (VWR #20104.243)
1 g Fuschine acide (Merck #1.05231.0025) in 100 ml distilled water, bring to boil and after cooling add 1 ml glacial acetic acid (VWR #20104.243)
Take 20 ml of each stock solution and make up to 400 ml with distilled water
► Aniline blue solution :
1 g aniline blue (Roth #4002.2) in 100 ml distilled water
bring to boil and after cooling add 1 ml glacial acetic acid (VWR #20104.243)
► Result
collagen: blue
nuclei: black
muscle, cytoplasm, keratin: red-purple