Staining Techniques. Staining Techniques. There are many dyes. Histochemical Stains: chemical reactions. Feulgen reaction -DNA

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1 Staining Techniques There are many dyes. Examples: Sudan black -Lipids Myelinated axons- blue ihcworld.com/imagegallery/displayimage.php?al... Weigert Stain -Reticular fibers Staining Techniques Histochemical Stains: chemical reactions Feulgen reaction -DNA Periodic Acid Shiff (PAS) page=ls&content=gallery&sub=feulgen -neutral and acidic polysaccharides - glycogen, mucous, basal laminae Intestinal Villus Goblet cells PAS stain 1

2 Staining Techniques Localization (staining) of an enzyme Staining Techniques AB + T ACETYL CHOLINESTERASE AT + B Acetylcholinesterase- neuromuscular junction AB + T provide substrate ENZYME AT + B generate visible product Other stains for ATPases, alkaline phosphatases, and others IMMUNOCYTOCHEMISTRY Use of antibodies to detect specific molecules (antigens) in a tissue Antibody binds to an antigen in the tissue. ANTIBODY ANTIGEN IMMUNOCYTOCHEMISTRY Direct Immunocytochemistry: a visible marker is directly attached to antibody binding the antigen The antibody is conjugated to visible marker.!fluorochrome!enzyme (HRP)!Electron dense molecule (ferritin, gold) Procedure: Fix the tissue Rinse with saline solution Incubate with conjugated antibody Rinse Mount on slide, view under microscope 2

3 DIRECT IMMUNOCYTOCHEMISTRY ADVANTAGES Specificity Less background staining INDIRECT IMMUNOCYTOCHEMISTRY! Primary antibody binds to the antigen. DISADVANTAGES Low sensitivity if the antigen is present in the tissue in low concentrations. Need to directly conjugate marker to antibody. INDIRECT IMMUNOCYTOCHEMISTRY! Primary antibody binds to the antigen.! Secondary antibody binds to the primary antibody.! Secondary antibody is conjugated to a visible marker. INDIRECT IMMUNOCYTOCHEMISTRY ADVANTAGES Amplification of the signal Can use labeled secondary with different primary antibodies Procedure: Fix the tissue Rinse Incubate -unlabeled primary antibody Rinse Incubate labeled secondary antibody Rinse Mount on slide, view under microscope DISADVANTAGES The nonspecific background may increase Takes longer to do Needs more reagents 3

4 LIMITATIONS OF IMMUNOCYTOCHEMISTRY Cross-reactivity Sensitivity Antigenicity -Frozen sections Types of antibodies used in immunocytochemistry Polyclonal antibodies -Several antibodies recognize >1 different epitopes on the same antigen Monoclonal antibodies -A single antibody recognizes only 1 epitope 4

5 ! Polyclonal antibodies!advantages: recognize more epitopes in tissue!disadvantages: less specificity Antibody against laminin Antibodies against different epitopes of myosin heavy chain! Monoclonal antibodies!advantages: more specific!disadvantages: reduced signal possible Representative myosin heavy chain (MHC) immunocytochemistry images of an emphysematous diaphragm after co-incubation with anti-laminin antibody and an antibody against one of the adult MHC isoforms. Antibodies (immunoglobulins) of specific species are used as antigens to generate secondary antibodies. ANTIGEN--> mouse antibody Rabbit anti-mouse IgG Goat anti-mouse IgG Donkey anti-rabbit IgM QUESTION: Dr. Reist is studying the distribution of two proteins, FasII and spectrin in neurons. She would like to label both molecules in the same sample using doublelabeling immunocytochemistry. She has these antibodies: Primary antibodies: Secondary antibodies: rabbit anti-fasii mouse anti-fasii goat anti-fasii rat anti-spectrin mouse anti-rabbit-fitc(fluorescein) donkey anti-rabbit-fitc rat anti-mouse-rh (rhodamine) goat anti-mouse-rh rabbit anti-spectrin rabbit anti-goat-rh donkey anti-spectrin What primary and secondary antibodies will successfully distinguish the distribution of FasII and spectrin in the same preparation? 5

6 AUTORADIOGRAPHY! Tissue with radiolabeled molecule! Cover with photo emulsion! Radiation activates silver -> silver grains! Develop and view In situ hybridization Labeled DNA or RNA probe Why? 6

7 In situ hybridization Labeled DNA or RNA probe Radioactive tag Digoxigenin Incubation with tissue Autoradiography or Immunocytochemistry www-bioc.rice.edu/bios576/immuno/immuno.html Whole mount in situ hybridization views on E10.5 mouse embryos with Phox2a (A), En1 (B), Uncx4.1 (C) and Lmx1b (D) RNA-probes. Juha PartanenInstitute of Biotechnology, P.O.Box 56, FI Univ. of Helsinki Fluorescence in situ hybridization of the all-human telomere probe, (T2AG3)n, to chromosome ends of the Pacific oyster (Crassostrea gigas). 7

8 FOUR TISSUES - Epithelial Tissue EPITHELIAL TISSUE -covers surfaces - Connective Tissue -lines cavities - Nervous Tissue -forms glands - Muscle Tissue EPITHELIAL TISSUE Functions: 1) protect against abrasion 2) physical barrier 3) transport 4) excretion/secretion 5) sensation CLASSIFICATION -Exhibit Polarity Free surface-lumen Apical Basal Lateral -Close cell apposition adhere to one another specialized cell junctions -Attached to a Basal Lamina (Basement Membrane) 8

9 CLASSIFICATION of EPITHELIA -Number of cell layers -Cell shape of surface cells -Specialization of apical surface CLASSIFICATION -Number of cell layers Simple Stratified Pseudostratified Squamous: flat width > height Simple squamous: Single cell layer Blood vesselsendothelium Body cavitymesothelium Kidney Lung Epithelial Cell Shape 9

10 CLASSIFICATION Squamous: flat Stratified squamous: >1 cell layers [Basal cells: stem cells for upper layers.] Skin (epidermis) Mouth & Esophagus Vagina Anal canal Keratinized Stratified Squamous Epithelia Apical surface has a hardened layer of dead cells rich in keratin intermediate filaments.!provides protection against abrasion and dessication (drying). Skin Anal Canal 10

11 CLASSIFICATION Cuboidal: height of cells = width Simple: (eg. thyroid, ovary, kidney tubules) Stratified: (sweat glands) 11

12 CLASSIFICATION Columnar: height of cells > width Simple: can be ciliated intestine gall bladder fallopian tubes HistoTip: When evaluating columnar epithelia, note position of nuclei. Simple columnar, nuclei appear aligned; Psuedostratified, nuclei are more randomly distributed. Simple columnar- intestine 12

13 CLASSIFICATION Columnar: Stratified: (ducts of exocrine glands) CLASSIFICATION Columnar: Pseudostratified: single cell layer that appears stratified; each cell in contact with basement membrane; frequently ciliated Trachea Urethra Transitional Epithelium: stratified, domed superficial cells, allows for extension; bladder 13

14 Neal Kalra 08 Histology Distended bladder Empty bladder 14