Histochemistry And Other Miscellaneous.docx

1.

Enhanced polymer one step staining

2.

Peroxidase – anti-peroxidase (PAP) technique

3.

Avidin biotin techniques

complex

(ABC)

4.

Direct immunofluorescence technique for solid tissue biopsies

5.

Indirect technique

6.

Frozen immunocytochemistry

7.

Frozen section immunofluorescence

8.

Direct immunoperoxidase method

9.

Paraffin wax section immunoperoxidase technique

immunofluorescence

section

HISTOCHEMISTRY AND OTHER MISCELLANEOUS PURPOSE A large number of primary antibody molecules and peroxidase enzymes are attached to a dextran polymer ‘backbone’, hence increasing the signal amplification and provide greater sensitivity. The method has had three major applications: (1)staining of tissues with high levels of endogenous peroxidase (2)double immunostaining in conjunction with peroxidase (3)staining of specific cell types that benefit from the bright red color of alkaline phosphatase substrates These methods rely on the affinity of the glycoprotein avidin for biotin •Biotin, a low molecular weight vitamin, is easily conjugated to antibodies and enzyme markers •Up to 150 biotin molecules can be attachedto one antibody molecule → increased sensitivity considered as gold standard method to detect deposits of tissue-bound autoantibodies&immune complexes Mainly used for the detection of autoantibodies in the patient’s serum, including the anti-nuclear antibofy(ANA), antimicrobial antibody(AMA), and liver-kidney microsomal antibody

Identify antigens in fresh frozen sections

Extensively used to detect antibodies, particularly for the diagnosis of glomerular disease in frozen sections of renal biopsies detects antigens in tissues Standard method among most laboratories, especially in combination with frozen section processing for immunofluorescence of renal and skin biopsies

RESULT OF STAINING immunostaining can be performed in less than 1.5 min. Antibodies are available for more than 20 important tissue and tumor markers. --Nuclei Blue --Sites of peroxidase activity Black/Brown

The result is a greater concentration of enzyme (three enzyme molecules to one avidin molecule) at the antigenic site and therefore an increase in signal intensity and sensitivity upon addition of substrate.

Apple-green fluorescence when fluorescein is used as fluorochrome Oramge-red fluorescence with rhodamic conjugates Homogenous (diffuse) pattern: Typically associated with SLE, mixed connective tissue disease, and drug-induced lupus. Speckled pattern: Associated with SLE, Sjögren syndrome, scleroderma, polymyositis, RA, and mixed connective tissue disease. Nucleolar pattern: Associated with scleroderma and polymyositis. Centromere (peripheral) pattern: Associated with scleroderma and CREST (Calcinosis, Raynaud syndrome, esophogeal dysmotility, sclerodactyly, telangiectasia). Sharper and more extensive p53 protein expression was observed in frozen section as compared to formalin fixed paraffin embedded sections. Fluorescence of antibodies

staining of the majority of the intranuclear inclusions Demonstation of intracytoplasmic antigens