Cutting and Staining Techniques PDF

CUTTING CRYOSTAT ROTARY MICROTOME ULTRA MICROTOME FOR FROZEN TISSUE FOR PARAFFIN FOR RESIN STEEL BLADE  CUTTING FROZEN TISSUE A CRANK IS...

CUTTING CRYOSTAT ROTARY MICROTOME ULTRA MICROTOME FOR FROZEN TISSUE FOR PARAFFIN FOR RESIN STEEL BLADE  CUTTING FROZEN TISSUE A CRANK IS TURNED TO BRING THE FROZEN TISSUE BLOCK FROZEN TISSUE, IS AFFIXED ON A SAMPLE HOLDER CLOSER TO THE INSIDE THE COLD ROOM BLADE THE BLADE CUTS CRYOSECTIONS (5-7 mm) SECTIONS ARE STICK ONTO GLASS SLIDES AND STORED AT -20 °C UNTIL PROCESSING  CUTTING PARAFFIN EMBEDDED TISSUE PARAFFIN INFILTRATED SAMPLE IS AFFIXED ON A SAMPLE HOLDER A CRANK IS TURNED TO BRING THE PARAFFIN BLOCK CLOSER TO THE BLADE SECTIONS ARE THE BLADE CUTS REMOVED FROM THE SECTIONS (5-7 mm) BLADE WITH A FORCEP AND PLACED IN A BATH OF HOT WATER (37 ° C) SECTIONS ARE MOUNTED ONTO GLASS SLIDES AND ALLOWED TO DRY OVERNIGHT AT ROOM TEMPERATURE OR 37°C ARE STORED UNTIL PROCESSING  CUTTING WITH ULTRAMICROTOME GLASS KNIFE BOATS GLASS KNIFE KNIFE Sections are placed on grids CONTROL UNIT TO CUT THIN SECTIONS: -750 nm (optical microscope) -50-80 nm (TEM)  STAINING -TO INCREASE THE CONTRAST OF CELLULAR OR TISSUE MORPHOLOGICAL COMPONENTS TO OBTAIN A BETTER MORPHOLOGICAL ANALYSIS -TO IDENTIFY AND LOCATE SPECIFIC CHEMICALS WITHIN A CELL OR TISSUE REAGENTS REACT WITH SPECIFIC CHEMICAL COMPOUNDS CONTAINED IN CELLS AND TISSUES IN ORDER - TO FORM COLORED PRODUCTS (IN THE CASE OF OPTICAL MICROSCOPY) - TO FORM ELECTRONDENSE PRODUCTS (IN THE CASE OF ELECTRON MICROSCOPY)  TISSUE STAINING FOR LIGHT MICROSCOPY BIOLOGICAL DYES - AROMATIC ORGANIC COMPOUNDS A DYE GIVES COLOR WITH ITS CHROMOGEN -GROUPS OF ATOMS ABSORBING CHROMOPHORE OR REFLECTING CERTAIN WAVELENGTHS OF THE VISIBLE SPECTRUM -THE COLOR OF DYE DEPEND ON CHROMOPHORE AND APPEARS IN THE COLOR OF REFLECTED LIGHT AUXOCHROME -GROUPS OF ATOMS WITH ELECTRIC + - CHARGE (OH,- NH4 , COO, SOH3-) -IMPART POSITIVE OR NEGATIVE CHARGE TO CHROMOGEN IONIZED CHOROMOGEN BIND WITH CELL CONSTITUENTS WITH OPPOSITE CHARGES TISSUE CONSTITUENTS REACT DIFFERENTLY TO DYES AND ARE DIFFERENTLY STAINED  DYES CAN BE CLASSIFIED IN ACID OR BASIC ACCORDING TO THE CHARGE OF THEIR AUXOCHROME ACID - ANIONIC BASIC - CATIONIC - AUXOCHROME: NEGATIVE CHARGE -AUXOCHROME: POSITIVE CHARGE - CHROMOGEN: NEGATIVE CHARGED -CHROMOGEN: POSITIVE CHARGE - BIND TO POSITIVELY CHARGED - BIND TO NEGATIVELY CHARGED CELL STRUCTURES - PROTEINS CELL STRUCTURES - NUCLEIC ACID - STAIN CYTOPLASM - STAIN NUCLEI ES. EOSIN, PICRIC ACID, ACID FUCHSIN ES. CRYSTAL VIOLET, METHHYLEN BLUE, BASIC FUCHSIN, SAFRANIN DYES CAN BE CLASSIFIED IN NATURAL OR ARTIFICAL NATURAL ANIMALS: ES. CARMINIO (NUCLEI) VEGETABLES: Es HEMATOXYLIN (NUCLEI) ARTIFICIAL EOSIN (CYTOPLASM) FUCHSIN, CRYSTAL VIOLET (NUCLEI)  STAINING METHODS SIMPLE STAINING: A SINGLE DYE IS USED TO EMPHASIZE PARTICULAR STRUCTURES IN THE SPECIMEN CELLS IN A TISSUE APPEAR TO BE THE SAME COLOR DIFFERENTIAL STAINING: ACID AND BASIC DYES - TO DISTINGUISH DIFFERENT CELLULAR COMPONENTS DOUBLE STAINING NUCLEI DARK BLUE (HEMATOXYLIN) CYTOPLASM IN PINK (EOSIN) TRIPLE AND QUADRUPLE STAINING NUCLEI IN PURPLE (HEMATOXYLIN) CYTOPLASM IN RED (ACID FUCHSIN) COLLAGEN (ANILINE BLUE) ERYTHROCYTES (PICRIC ACID)  CHEMICAL - PHYSICAL STAINING SUBSTRATE AND DYE HAVE OPPOSITE CHARGES CHEMICAL BONDS ARE FORMED BETWEEN THE COLORED AND TISSUE MOLECULES HEMATOXYLIN–EOSIN STAINING EOSIN (ACIDIC DYE) CYTOPLASM HEMATOXYLIN (BASIC DYE)-NUCLEI  H&E FOR MORPHOLOGICAL EXAMINATION (TISSUE BIOPSY) CANCER MYOPATHIES DIAGNOSIS DIAGNOSIS EFFECTS OF THERAPY ON TUMORS -H&E-stained sections of dissected thymoma tumors from the control group (A) show the intact tumor cell structure. Cells have distinct nuclei with a thin uniform cytoplasmic rim around each individual cell. -Tumors treated with chemotherapy show nonuniform thickened rim of cytoplasm around the damaged nuclei (B). -Radiotherapy-treated tumor cells show distinct damage of individual tumor cell nuclei with distortion of the cell membrane (C). Damaged cells are indicated by arrows.  CHEMICAL STAINING CHEMICAL REACTIONS BETWEEN TISSUE MOLECULES AND NON COLORED REAGENTS FORMATION OF COLORED COMPOUNDS P.A.S. (GLYCOGEN) PERIODIC ACID OXIDIZES GLYCOLIC GROUPS ALDEHYDIC GROUPS ARE FORMED CHOH-CHOH CHO-CHO CHO-CHO + FUCSIN-SULFUR ACID CHANGES ALDEHYDIC GROUPS INTO AN INSOLUBLE PINK STAINED COMPOUND  PHYSICAL STAINING COLORED SUBSTANCES DISSOLVE IN OR DEPOSIT ON SPECIFIC TISSUE STRUCTURES NO CHEMICAL BONDS ARE FORMED OIL RED O O.R.O (LIPIDS) IDENTIFIES NEUTRAL LIPIDS AND FATTY ACIDS IN TISSUES (RED DROPLETS) THE STAINING IS PERFORMED ON FRESH SAMPLES, AS ALCOHOL FIXATION REMOVES MOST LIPIDS USED TO OBSERVE CHANGES IN THE LIPID METABOLISM: EXCESSIVE ACCUMULATION OF LIPIDS IS THE KEY FEATURE OF SEVERAL METABOLIC EVENTS AND / OR DISEASES SILVER IMPREGNATION (NEURONS AND THEIR PROCESSES) - SILVER DEPOSITION ON BIOLOGICAL STRUCTURES -REACTION BETWEEN SILVER SALT + REDUCING SUBSTANCE (ammoniacal solution) -RELEASE OF METALLIC SILVER THAT DEPOSITS ON NEURONS  STAINING PROCEDURE DYE SOLUTIONS ARE OFTEN DISSOLVED IN WATER TO ALLOW COLOURANT TO PENETRATE INTO THE TISSUE: -ELIMINATE THE PARAFFIN FROM TISSUE WITH XYLENE -REHYDRATE TISSUE WITH A DECREASING ALCOHOL SCALE (100 °, 90 °, 70 °, 50°) -COLORING MOUNTING OF SLIDES MOUNTINGS ARE HYDROPHOBIC DEHYDRATED BY USING GRADED ETHANOL SOLUTIONS MOUNT WITH MOUNTING MEDIA (EUKITT) COVERSLIP  DOUBLE - CHEMICAL - PHYSICAL STAINING HEMATOXYLIN – EOSIN (NUCLEUS-CYTOPLASM) XYLENE (DEPARAFFINIZATION) 5 min. ETHANOL 100% 3 min. ETHANOL 90% 3 min ETHANOL 70% 3 min ETHANOL 50% 3 min DISTILLED H2O 3 min HEMATOXYLIN 5 min TAP WATER 5 min EOSIN 5 min TAP WATER 5 min DISTILLED H2O rapid wash ETHANOL 50% 3 min ETHANOL 70% 3 min ETHANOL 90% 3 min ETHANOL 100% 3 min XYLENE 5 min MOUNT WITH EUKITT AND COVERSLIP QUADRUPLE - CHEMICAL - PHYSICAL STAINING MASSON TRICHROME BRING SECTION TO DISTILLED WATER PUT ON THE SECTION 6 DROPS OF REAGENT A AND 6 DROPS OF REAGENT B 10 min DRAIN THE SLIDE AND PUT ON SECTION 10 DROPS OF REAGENT C 4 min WASH QUICKLY IN DISTILLED WATER 3-4 sec PUT ON SECTION 10 DROPS OF REAGENT D 4 min WASH QUICKLY IN DISTILLED WATER PUT ON SECTION 10 DROPS OF REAGENT E 10 min DRAIN THE SLIDE AND PUT ON SECTION 10 DROPS OF REAGENT F 5 min WASH IN DISTILLED WATER ALCOHOL 70° 1 min ALCOHOL 90° 1 min ALCOHOL 100° 1 min XYLENE 1 min MOUNT WITH EUKITT AND COVERSLIP A+B Weigert’s iron hematoxylin for nuclei E Phosphomolybdic acid solution C Picric acid alcohlic solution for erythrocytes F Aniline blue for connective tissue D Ponceau acid fuchsin for cytoplasm  CHEMICAL STAINING P.A.S. (PERIODIC ACID OF SCHIFF- GLYCOGEN) INCUBATE WITH PERIODIC ACID OF SCHIFF (A) 10 min RINSE WITH DISTILLED WATER 4 TIMES INCUBATE WITH SCHIFF REAGENT (B) 15 min RINSE WITH DISTILLED WATER 4 TIMES POTASSIUM METHABISULPHITE (C) 2 min FIXATIVE SOLUTION (D) 2 min RINSE WITH DISTILLED WATER 5 min ALCOHOL 70° 1 min ALCOHOL 90° 1 min ALCOHOL 100° 1 min MOUNT WITH EUKITT AND COVERSLIP  PHYSICAL STAINING SILVER IMPREGNATION XYLOL 5 min ETHANOL 100 ° 3 min ETHANOL 90 ° 3 min ETHANOL 70 ° 3 min DISTILLED WATER 5 min 5 DROPS REAGENT A + 5 DROPS REAGENT B (OXIDATION WITH POTASSIUM PERMANGANATE) 5 min WASH WITH DISTILLATE 10 DROPS REAGENT C (OXALIC ACID) 3 min WASH WITH DISTILLATE 10 DROPS OF REAGENT D (IRON AMMONIUM SULFATE) 3 min 2 WASHES IN DISTILLED WATER 10 DROPS OF REAGENT E (AMMONIA SOLUTION WITH AG IN WATER-SOLUBLE OXIDE COMPLEX) 3 min WASHING WITH DISTILLED WATER 10 DROPS OF REAGENT F (FORMALDEHYDE IS A REDUCING AGENT AND FREE IONS Ag) 5 min 2 WASHES WITH DISTILLED WATER 10 DROPS OF REAGENT G (SODIUM HYPOSULPHITE TO ELIMINATE EXCESS IONS Ag) 5 min WASHING IN SOURCE WATER ETHANOL 70 ° 3 min ETHANOL 90 ° 3 min ETHANOL 100 ° 3 min XYLOL 3 min EUKITT