histology Flashcards
how do we get antibodies for use in IHT
immunise a rabbit with desired antigen
harvest blood palsma
collection with be specific to chosen antigen
specificity of polyclonal antibodies
poor
how are monoclonal antibodes made
clone of plasma cells from spleen
cell culture poducig clonal antidies coupled with cancer cell line, unlimited suply of Ab
use of horseradish perxoidase
natural enzym from the root of horse radish plant
in the presense of hydrogen peroxidase it causes oxidation to DAB
led to development of PAP ad APAAP methods
PAP method
peroxidase anti peroxidase method
first Ab bound to antige
then bridge AB added for the PAP complex
ABC method
Advin-biotin complex method
involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase. The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.
advin strepbiotin peroxidase method
more sensitive method
the first layer is unlabeled primary antibody.
The second layer is secondary antibody.
The third layer is Enzyme-Streptavidin conjugates.
The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products.
polymer methods
binding of a large number of enzyme molecules (horseradish peroxidase or alkaline phosphatase) to a secondary antibody via the dextran backbone. The benefits are many, including increased sensitivity, minimized non-specific background staining and a reduction in the total number of assay steps
human tissue act 2004`
removal,storage and disposal of human tissues needs to be regulated
fixation of tissues
formaldehyde 4%
enbles morphology to be studies and kept is life like state
cut up
represenative sample of a specimin is taken by a a pathologist and placed in casette
what is key in embedding
IRENTATION OF SAMPLES
How does H and E work
Nuclei stained blue by haematoxylin through a mordant bond followed by counter stadin with eosin for pink coloiur
dark field microscope
condesor blocks out bright light and illuminates the sample by pblique light chancing contract brining out fine detaisls better
phase contrast
living samples
light split above sample making an image where structures are darker than background
