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