histological techniques Flashcards
neurohistological techniques
nissl’s method: basic dyes- toluine blue, methylene blue and thionine (stains nissl’s body, nuceli+nucleoli
myelin sheath-luxol blue (phospholipid) +weigert method (mordant, uses chromium salt to stain myelin sheath)
neutral lipids, cholesterol esters (Nile blue sulfate) unsaturated lipids (osmium tetroxide) reduction of osmium by unsaturated FA.
structures of neuron
dendritic spine-silver impregnantion
neurofibril (IF)- silver impregnantion
neuron cells have conspicuous nucleoli= why cyto eosinophilic
oil red+ sudan black stains lipids- myelin
MAJOR BASIC PROTEIN also
types of techniques to collect sample+preparation of slides
biopsy/necropsy
technique
1) excision-cutt off tissue w sample. in surgery or in biopsy.
2) needle biopsy-take sample from organs (liver/pancreas, bone marrow, thyroid, breast) or fine needle biopsy to get lumps from under skin
3) curretage- microabression/scraping off. done in uterus mucosa (endometrium) uses currete 30cm long, to check menstrual cycle
4) endoscopic- special tool goes through cavity to see organs
5) trepanobiopsy/exfoliative cytology- extracting smears from vagina by shedding.
preparation of slides
1) Fixative- important bc preserves tissue by cross-linking degenerative inactive enzymes. cause autolysis +kil pathogenic microorganism
requirement: cut to small fragments (faster); maintain stainability+make sure its preserved
- physical: heat, microwave or freezing (lipids bc fixative often dissolves a bit of lipid and N(l) prevents that. use cryostat to cut those samples.
- chemical fixation(corss link proteins) submerge tissue in fixative-FORMALDEHYDE (37%-40%)=formalyn most used. GLUTEROALDEHYDE another fixative. formaldehyde used w baker's fluid (CaCl)-specific to lipids Not suitable for fixation of bloody organs and lipids; picric acid hemolysis blood leading to hard clots in tissues, lipids or anything before celloidin embedding.
Before use 5ml glacial acetic acid is added per 100ml fixative. Fixation takes 24hrs, and should be washed in 80% ethanol afterwards.
picric acid w two fluids- Bounins fluid and Gendre’s fluid (picric (immersed w methanol not aq sol)= specific to polysaccharides
Mercuric acid: uses ZUZA fluid (NaCl)+ Zenker’s fluid (K dichormate) =specific to polysaccharide fixation
2)embedding-submerse in medium to facilitate section. Water soluble-gelatin+synthetic resin. insoluble- paraffin wax (common; hydrocarbons seen in light microscope)+ celoidine
3) dehydration remove water using ethanol (70%-80-90-94-100; in embryonic 30)
4) clearing remove OH, needs to be misceble w paraffin+OH (benzene, tuloidine, methol or xylene)
5)infiltration/impregneation place tissue inside paraffin wax. put in oven 52-60C
6) embedding immersed in paraffin so left out to air dry+harden. then microtome used to cut it 4-10um. then immersed in warm water and mounted on slide w albumin adhesive.
methods for staining + diff types of stianing
deparaffinization- last step was drying so
1) remove paraffin: 2X xylene +2X ethanol (96,100%) +wash in distilled water
2) staining: hemotoxylin- meyers. rinse w tap water to neutralize pH. differentiatiate w acid ethanol. rinse again. and stain w eosin 0.5%sol, wash in h20. differentiate w 80% ethanol
3) dehyfration- remove h20 by adding ethanol (2X). add xylene m
4) clearing- remove ethanol using 2X xylene
5) mounting- embedded medium synthetic reason or Canada baiasain
Nuclei = blue/purple
Cytoplasm = pink shades
Collagen Fibres = pinkish red
Muscles = deep pinkish red
Erythrocytes = reddish/pinkish/orangey
diff types
- Progressive – dye is taken up by the tissue and it is not removed
- Regressive – Overstaining and remove excess dye
- Successive – Staining step by step w different dyes
- Simultaneous – dyes dissolved in one bath.
- Vital Stain – stain that can be applied on living cells without killing them
- Supra Vital staining – living cells that have been removed from an organism
- Intra Vital staining – Injecting or introducing the stain into the body
- Direct vs with mordant (aluminum, tungsten or ferric salts) – mordant enhance staining ability of some dyes, e.g. alum or iron haematoxylins.
immunohistochemistry
reactions of antibodies+antigens. antibodies produced by injecting one animal protein to another (human to mouse). causes antibody to be produced since aa seq for that system is recognized as foreign, creating antibodies against this protein.
- Tagged using fluorescent compounds: peroxidase, alkaline phosphate e- dense gold particles- for TEM.
two types of classifications :
1) monoclonal- antibody produced w one b cell. can only recognize one epitope. (b cell recognizes foreign particle, isolated and cloned to make more antibodies)
2)polyclonal: recognize more than one epitope. made by multiple b cell, therefore vary in specificity (not all the exact same antibody)
Methods: direct vs indirect
- direct- labeled+ tagged antibody incubated w tissue. wash it out to remove any antibodies that did not bind to antigen.
- indirect: primary antibody not tagged, binds to antigen. antibody from another animal inserted+ binds to primary antibody causes it to become more sensitive, amplifying signal.
immunofluorescent dyes
dna-blue, lysosome-green, endocytitic-red
visualize specific mol
mol interact specifically w other mol
phalloidin- mushrooms- interacts w actin
protein A- bacteria- interacts w Fc region at immunoglobulin. antibody and tail has fc interacts w fc site on proteins its trying to bind to, Lectin-glocoprotein-plants- affinitive to carbs
staining table
blue trichrome+azan CT (collagen)=blue
van geison red/majenta
differentiate between cyto= green trichrome (yellow-ish) + van geison (more pale yellow) everything else salmon pink
nuclei- yellow trichrome+hemotoxylene (dark blue); green+blue trichrome and van geinson (brown). Azan (red)
muscles all red except van geinson (yellow +erythrocyte as well). azan (dark red+erythrocyte violet)
erythrocyte all red expect green trichrome(yellow) , azan (violet) and van geinson(yellow)
composition
basic dye:
Hemotoxylin aluminium (mordant-doesn’t actually stain enhance other structures stainability) and eosin
yellow trichrome: Hemotoxylin aluminium+ saffron+erythrosin
green trichrome: weigert iron hematoxylin+ acid fuchsin+light green+orange g+ ponceau de xylidine
blue trichrome: hematoxyline+ acid fuchsin+aniline blue+ponceau de xylidine
(van geison) :weigert’s hematoxylin +picric acid
azan-azocarmine+ aniline blue+ orange D
perichondrium- EOSINOPHILIC (fibrous layer)
elastic: recorsinfuchsin, aldehydofuchsin+orcein+ weighert
adipocytes (in inclusion material ex) OIL RED/SUDAN BLACK/
heparinocyte (mast) ALCIAN BLUE/ PAS+
(periodic acidic schiff)
mucous cell- PAS+ and ALCIAN BLUE (just like mast)
serous cell- PAS- +eosinophilic but cyto is basophilic (rer+GA)
Azurophilic granules AZURE A
cartilage matrix ALCIAN BLUE, NUCLEAR RED(acidic) +H&E
Histochem
DNA-Fuelgen’s rection - deoxyribose sugars are hydrolysed by HCl
RNA METHYLENE/TOLUIDINE BLUE
lysosomes-TOLUIDINE BLUE, in endothelial cells stains FLOURESCENT
enzyme histochem
specific enzymatic reactions to localize structures
to preserve the enzyme =mildly fixated/not at all. freezing useful
1) submersing tissue into sol w substrate of enzyme we want. React + product marked/labelled
2) product becomes insoluble, visible by TEM/optical microscope, precipitate over enzymatic reaction (what we see to identify)
3) 3 types of enzymes:
phosphatase(remove PO4 from phosphorised mol)
dehydrogenase (remove H from substrate and transfer to another mol-needs mol that receives protein and precipitate as insoluble coloured compound; often used to find mitochondria)
peroxidase (incubate tissue in sol w 3-3-diaminobenzidine. in H2O2 oxidise=precipitate) . precipitate is insoluble brown e- dense
liver-pas+ and hemotoxylin
