Visualization Techniques - Heat-sensitive (destroyed in
processing)
- Methods to elucidate structures and • Analyte- Enzyme inside cells & tissues
chemical components of cells & tissues
o peroxidase, lipases,
• Staining- application of dyes/stain
phosphatases
• Tissues have varying affinities
• Localize enzyme: mixed with substrate
• Stain – can be erased (reagent)
• Dye – imparts permanent colors; 2 - Substrate- what the enzyme will
components: consume
o Chromophore – impermanent • Reaction cannot be seen without the
color color: add developer
o Auxo chrome – dyeing property; - Developer: chromogen
allows color to retain o Has different types; will depend
• Hematoxylin- basic (will stain the acidic on the enzyme to be used/
nucleus), anion, nuclear stain; blue-black procedure (red, brown)
o Harris Hematoxylin • Tissue: add reagent + substrate +
- Routinely used Chromogen
- Indirect stain – requires o Colored portions indicate the
mordants enzyme.
o Mordants are link/bridges • Appropriate Specimen: Frozen sections
between the tissue and dye - Viable for enzyme histochemistry
- binds them both to form - Immersed in liquid nitrogen: freeze
complexes. - Sectioned in Freezing Microtome
• Eosin – acidic, cation, cytoplasmic stain, o Processed tissue: already
red-pink undergone different processes
o Eosin Y – routinely-used (red,
orange, pinkish) IMMUNOHISTOCHEMISTRY
o Eosin S, Erythromycin B • “Immuno” = antigen (Ag) & antibody (Ab)
reaction
Groups of Staining • Antigen – any foreign substance that will
HISTOLOGIC STAINING react with an antibody (proteins, lipids,
- Staining of morphology or different carbohdrates or mixture; there are
structures of cells and tissues antigens in our body)
- Most stains fall under this group • Antibody– proteins produced in
o H&E Staining Technique response to a specific antigen
o Immunoglobulins,
HISTOCHEMICAL STAINING gammaglobulins
- Demonstration of chemical components • Immune complex = specific binding of
• Perl’s Prussian Blue – for iron Ag & Ab
• Periodic Acid Schiff (PAS) – for glycogen • Analyte- what we are looking for in a
(stored energy of tissue) tissue
• Enzyme Histochemistry o Tissue antigens- phenotypic
- Demonstration/localization of enzymes markers
- Biochemical catalysts; speed up • Reagent- corresponding antibody
reactions o IgG Antibody
- Mostly proteins
- Found in tissues
o others: IgA, IgM, IgD, IgE • Applications:
(GAMDE) o Detection of tumor markers (not for
o IgG can be: diagnosis, but for monitoring the
o Monoclonal tumors)
- comes from one type of source o Microbiology: detection of bacteria
- more specific: react w/ 1 type of
antigen AUTORADIOGRAPHY
- Generated from mice • Detection of newly synthesized
o Polyclonal macromolecule .
- less specific: can bind with o DNA, RNA, protein, carbohydrates
different antigens • Process:
- generated from rabbits o Expose to radiation
• Labels – allow the formation of the o Application of radioisotopes
immune complex to be seen under the o Addition of microdetectors (silver
microscope; attach to the antibodies bromide) -> colored reaction
• Labelled Immunoassays (assays = tests) o Appearance of macromolecules as
o Enzyme labels (Substrate + silver grains
Chromogen)
- To observe: colored product; Cell & Tissue
ordinary microscope to see • Culture media (agar)- to grow bacteria
§ Alkaline phosphatase
• Culture Unculturable Organisms
§ Horse radish peroxidase o Viruses, Chlamydia (intracellular),
o Fluorochrome Rickettsia
- To observe: Fluorescence - Do not grow on artificial environment
microscope - Use viable cells and tissues
- Fluorescent dyes; FITC
• Direct Method
- Primary antibody is already labelled and
specific to the antigen: tissue antigen + • Primary Cell Culture
labelled antibody • Derived from parent tissue
o Direct Enzyme Immunoassay - Finite
o Direct Immunofluorescence o Can reach the point where it won’t
• Indirect Method be available
- Two or more antibodies o Human embryonic kidney, rabbit
- Tissue Antigen + Primary Antibody + kidney
Labelled Anti-IgG Antibody • Immortal Cell Line
o Anti-IgG Antibody: will not bind to o Infinite passage: grow indefinitely
antigen because occupied; not o Immortal because of cancer cells
specific to Ag but to Ab; specificity is ⁃ when combined with normal cells,
in the complex they are immortal and continue
- more sensitive growing
- more amplified result o HeLa Cell Line, Hep-2
- more commonly used o Henrietta Lacks died of cervical
cancer and a sample of cervical
cancer cells was taken.
PROCESS o Basis of separation of proteins: affinity
• Inoculate culture- introduce organisms to Anodes (+)
to culture o Faster migration = smaller protein
• Visible growth: cytopathic effects; no (more anodal)
colonies o Higher protein components (more
• Changes in structure of tissue culture anodal)
o Multinucleation o Less protein component: cathode
o Nuclear inclusions
o Cytoplasmic inclusions
• Stain with H&E to view
• IMMUNOBLOT TECHNIQUE
o Western Blot = protein
HYRIBIDZATION TECHNIQUE - Confirmation test for HIV
• Molecular Techniques – method of o Northern Blot = RNA
binding of single strands of nucleic o Southern Blot = DNA
acids to its complementary strand:
probe
o In order to be detected: more
similar, more complementary SAMPLE PROCESS
o Single strand of nucleic acid + Probe
= Hybrid • VICTORY Protein
• Perform Electrophoresis to separate
• Fluorescent in-situ hybridization (FISH) protein into fragments.
o Labels: Fluorescent dyes • Blot to nitrocellulose paper
o In-situ (on-site): binding happened in • Add reagents as detectors of proteins of
the tissue interest
o Application: Genetics, cancer-screening o Reagents are usually antibodies:
Anti-R protein
• Gene Splicing • Since the antibodies will only bind to
o Portion of gene is inserted into a specific antigens on the protein, they
plasmid will be the only ones visualized. (only R
o Plasmid – extrachromosomal / will be visualized)
extranuclear genes • Upon identification and isolation of
o Process: proteins of interest,
o Restriction enzyme breaks up • perform Polymerase Chain Reaction
plasmid
o Gene of interest is incorporated: • Polymerase Chain Reaction
Recombinant plasmid o Function: Gene amplification
o Process:
• Electrophoresis o Denaturation
o Application of electric current to o Annealing
separate proteins into fragments o Elongation / Extension
o Usually performed in an agarose gel
o After proteins; at pH 8.6: negatively-
charged
o Add proteins on the cathode (-) side
