- Week 19: Histochemistry/Immunohistochemistry - Tissue - Cells - Cytology samples may be produced by exfoliation, abrasion (e.g. by brushing or scraping) or aspiration (e.g. using a f i ne needle). - Spatulas used for obtaining cervical cells - Specein dissected -Fixation,dehydration,clearing,infiltration - Embedding - Microtomy - Histochemistry: used for studying chemistry of tissues and cells - Haematoxylin and eosin(H&E) - Millers Elastic Van Gieson (EVG) - dye forms hydrogen bonds with elastin f i bres, Mallory bleach to sensitise f i bres, followed by staining with Millers elastic stain, Van Gieson stain is made up by mixing 0.5mls of 1% Acid fuchsin with 4.5mls of Saturated aqueous Picric acid. - RETIC stain - silver technique, Sensitise reticulin f i bres by oxidation, Deposition of silver ions, Reduction of silver ions, Tone with gold chloride to increase contrast, Counterstain - Periodic Acid Schif f (PAS) - stains glycogen, mucin, mucoprotein, glycoprotein, as well as fungi, predigestion step with amylase will remove staining for glycogen, PAS is useful for outlining tissue structures -- basement membranes, capsules, blood vessels, etc. Periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta colour. - Alcian Blue - acid mucus substances and acidic mucins. - Toluidine Blue - Metachromatic stain, blue stain that stains specif i c components of tissues a purple colour - Immunohistochemistry: labelled antigen-antibody reactions - Procedure: f i xing, sectioning, antigen retrieval, blocking, incubation with primary AB, incubation with secondary AB, introduction of substate, nuclei counterstaining and mounting
- Proteolytic antigen retrieval - Buf fers for washing ie PBS or TBS - Blocking: background staining can be specif i c or non-specif i c - Good antibody: af f i nity to antigen, binding property/avidity, concentration (titration) - Polyclonal antibodies: derived from dif f erent types of immune cells, af f i nity to different epitopes of antigen, easy to produce/cheaper, less specif i c, animals used - Direct, indirect, - substrate - Nuclear counter staining - Immunomarkers