Microwave procedure for Masson’s trichrome kit (HT15-1KT, Sigma-Aldrich, Germany) was used for infarct size estimation with the omission of counter staining with hematoxylin. Skin was the positive control.
5μm slides were prepared from paraffin blocks and mounted on Poly L-lysine coated slides. These slides were dewaxed in xylene and hydrated with decreasing gradations of alcohol and placed in a Coplin jar filled with Bouin’s solution (500ml distilled water was saturated with picric acid and then filtered 167ml formaldehyde and 33.3ml glacial acetic acid were then added to it). This was microwaved for 15seconds followed by a 5mins incubation in fume hood. Slides were then washed with tap water until yellow color on the slides disappeared. Slides were then stained with Biebrich Scarlet-Acid Fuchsin and microwaved for 12seconds, followed by a two minute incubation and then placed in Phosphotungstic/Phosphomolybdic acid (10ml Phosphotungstic acid and 10ml Phosphomolybdic acid is mixed in 20ml deionized water) and microwaved for 12seconds followed by 5mins incubation. Slides were then placed in Analine Blue solution, microwaved for nine seconds, followed by a one minute incubation. After several washes of deionized water, slides were placed in 1% acetic acid (1N acetic acid) for 45 seconds and washed with deionized water for two mins. This was followed by sequential dehydration with alcohol and clearing with two changes of xylene. Slides were then mounted with DPX.
5μm slides were prepared from paraffin blocks and mounted on Poly L-lysine coated slides. These slides were dewaxed in xylene and hydrated with decreasing gradations of alcohol and placed in a Coplin jar filled with Bouin’s solution (500ml distilled water was saturated with picric acid and then filtered 167ml formaldehyde and 33.3ml glacial acetic acid were then added to it). This was microwaved for 15seconds followed by a 5mins incubation in fume hood. Slides were then washed with tap water until yellow color on the slides disappeared. Slides were then stained with Biebrich Scarlet-Acid Fuchsin and microwaved for 12seconds, followed by a two minute incubation and then placed in Phosphotungstic/Phosphomolybdic acid (10ml Phosphotungstic acid and 10ml Phosphomolybdic acid is mixed in 20ml deionized water) and microwaved for 12seconds followed by 5mins incubation. Slides were then placed in Analine Blue solution, microwaved for nine seconds, followed by a one minute incubation. After several washes of deionized water, slides were placed in 1% acetic acid (1N acetic acid) for 45 seconds and washed with deionized water for two mins. This was followed by sequential dehydration with alcohol and clearing with two changes of xylene. Slides were then mounted with DPX.
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