Staining with semichon's acetocarmine
Primary Fixation
Fix specimens or tissues and affix them to slides or coverslips. AFA is the preferred fixative. Note that Semichon's is itself a fixative originally designed to simultaneously fix and stain wet smears. We move our coverslips from columbia jars to ceramic staining racks for processing. All stains and solutions are kept in screw top jars. We remove the lids as needed and move the racks from jar to jar and then tighten down the lids for storage.
Fixative Rinse
Rinse out excess fixative in 70% Ethanol for 3 minutes. Specimens that have been stored in 70% ethanol can go directly to staining.
Staining
Semichon's acetocarmine is a regressive stain, that is, specimens are overstained and then destained to remove excess stain and differentiate structures.Stain in Semichon's Acid Carmine for 2-3 minutes. Large gregarines may need to stain for up to 25 minutes.
Destaining
Rinse out excess stain in 70% Ethanol for 3 minutes. Destain in 1% Acid Alcohol. Time varies from 5 seconds to 5 minutes depending on species and size. Remove excess background stain by dipping in 5% Acid Alcohol for 1-2 seconds. Rinse in Alkaline Alcohol for 3 minutes to neutralize destain acids and set the final stain tone and color.
Dehydration
Dehydrate in a graded ethanol series: 95% (5 minutes); 100% (3 changes, 3-5 minutes each).
Clearing
Clear in a graded xylene series: 50% xylene/50% Absolute alcohol (5 minutes); 100% xylene (3 changes, 3-5 minutes each).
Mounting
Mount in warm (37-40 C) Damar Balsam.
