Demonstration(nucleic acid & lipids)

Demonstration(nucleic acid & lipids)

1. Amino acids/Nucleic acids

   • hydrolysis of nuclei acid
     • Bases
       • -no satisfactory histochemical method
     • PO4
       • -ionize at pH 1.5-2 -negative charge -basic dye staining by salt-linkages and H-bonding
     • Carbohydrates
       • useful for DNA demonstration
   • Methods
     • 3.Acridine orange
       • -Fluorochrome dye(basic dye) -used in smears or cytostat section -stain DNA & RNA
       • Result: -DNA:fluoresces green -RNA:fluoresces orange-red
       • fixation
         • -short fixation in acetic acid ethanol or 70% alcohol(except bouin)
     • 2. Methyl Green Pyronin
       • combine two basic dyes for differential staining of RNA and DNA.
       • the critical factor can affect the result: pH: 1. low pH(1.5); favours pyronin staining 2.high pH (9):favors MG staining 3.at pH 4.8:differential staining-DNA green(specific), RNA red(non specific ,mucous cells also stained red)
       • specificity
         • -MG;highly specific for DNA,not for RNA as they are protein bound. -Pyronin:it is small size, can stain RNA, depolymerized DNA, and mucous cells
       • factors affect result
         • fixation required
           • 1.alcoholic fixative -avoid formalydehyde dichromate(e.g. zenker/helly) -delay fixation or  prolog fixation in HCHO,cause loss of MG colour
         • Dye sample
           • -pyronin Y/G prefer to pyronin B(less diffuse and better differentiation) -MG impurities:from chloroform extraction of methyl violet
       • Applications
         • 1.histologic evaluation of immunologic disorders
         • 2. detection of RNA in: -plasma cells -tumours -plasmacytoid lymphocytes -viral inclusion bodies
     • 4.Gallocyanic chrome alum
       • -enzyme extraction:can stain one molecule on one section
     • 1. Feulgen
       • 1. acid hydrolysis(1M HCl@ 60) of DNA -to break the purine-deoxyribose bond: break the sugar linkage of polymeric bonding; ruptures the glycoside linkages between sugars and bases, yield aldehyde groups
       • 2.demonstration by schiff's reagent: result: aldehyde recolor schiff's reagent and produce magenta colour in the nuclear chromatin
       • restriction
         • cannot demonstrate RNA:it is removed by acid hydrolysis(depolymerization of RNA upon acid hydrolysis) -Purine bond and PO4 bond unaffected)
       • fixative required
         • 1.buffered formalin or acid fixatives (except BOUIN: over oxidation of nuclei acid)
       • Factors may affect result
         • 1.fixative:constitute/ nature 2.time of hydrolysis 3.temperature of hydrolysis 4.nature of specimen 5.early post-mortem changes 6.penetration of fixative 7.acid-pretreatment
       • Applications
         • 1.confirm/exclude DNA content of a basophilic inclusion 2.provide the distribution of DNA in various cells: due to precise site of DNA, semi-quantitative 3.nuclear sex studies-Barr bodies

2. Lipids**

   • Free lipid droplets destroyed by chloroform, acetone and during processing: phospholipids and lipofuchsin can bind to other tissue element and resist paraffin processing(can be demonstrated)
   • classification
     • a.simple lipid
       • properties: 1.esters of fatty acids and alcohols 2.Fats:natural ester of glycerol with saturated and unsaturated fatty acids 3.oil:similar to fats,liquid at RT
     • b. compound lipids
       • properties: 1.e.g. phospholipids, glycolipids 2.consist of a fatty acid, alcohol and one or more other groups(phosphorus/nitrogen)
     • c. derived lipids
       • 1.Fatty acids originate from simple and compound lipids by means of hydrolysis 2.e.g. cholesterol,sex hormone,adrenocortical hormones
   • fixation
     • 1. no ideal fixatives, unfixed frozen sections
     • special fixative: (i)osmium tetroxide and chromic acid- can fix lipids but will alter chemical reactivity of lipids  (ii)formal calcium-for lipid histochemistry (iii)formaldehyde-fix minority of lipid(neutral fat)                -cause damage after prolonged fixation
   • staining method
     • 2. histochemical
       • demonstrate individual lipid by specific method
     • 1.histophysical
       • 1.without dyes or 2. with dyes
       • Birefrigent
         • crystalline lipids are birefrigence:Maltese cross is found in cholesterol esters of lipids -fatty droplets no birefrigent (called :isotropic)
         • usage??
           • 1.distinguish between glycerol ester(cholesteryl esters)- neutral fats(mainly triglycerides:non birefrigence) -can't be identified solely by their optical properties
       • Autofluorescent
         • results: 1.ceroids;yellow 2.lipofuscin: yellow 3.vitamin A:green
       • step 1. Lipid extraction
         • specific of staining technique
         • step 2. Staining
           • oil soluble dyes
             • Properties/features 1.Lysochromes 2.Most are azo-dyes
             • oil red O
               • The solvent included: 1. Triethyl phosphate:stable,no ppt, no effect on lipid 2. 70% alcohol 3. 60% isopropanol 4. ethylene glycol 5.Propylene glycol
             • Bromine-Sudan Black
               • 1. simple sensitive method for detecting all main lipid classes. 2.only acetone resistant phospholipids are demonstrated
               • The role of bromine: 1. render unsaturated lipids insoluble in organic solvents 2. converts cholesterol to cholesterol bromide esters: lower MT,oily at RT, and permeable to Sudan dye.
             • Sudan Black B
         • Methods
           • a. delipidization
             • lipids can be totally extracted with chloroform and ethanol
           • b. keilig extraction(acetone used)
             • acetone used with different temperature: a)cold acetone extract:simple lipid, cholesterol, triglycerides b)hot acetone:glycolipids 
           • c. acetone extraction
             • properties: -acetone selectively remove hydrophobic lipid
             • process??
               • treat dried sections with anhydrous acetone(20 mins): 1.remove fat and cholesterol 2.acetone insoluble phospholipid intact(retained) 3.can also remove significant amount of phosphoglyceride
         • technical concern
           • 1.lipids seldom in pure form:they can dissolve in other lipids 2.bind with other tissue components and resists extraction(e.g. glycolipid, lipoproteins)
           • 3.No ideal solvent to extract one lipid and not affect others 4.require different solvents to extract different lipids under different conditions