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Role of microwave heating in antigen retrieval in cryosections of formalin-fixed tissues


Brzica, Hrvoje; Breljak, Davorka; Vrhovac, Ivana; Sabolić, Ivan
Role of microwave heating in antigen retrieval in cryosections of formalin-fixed tissues // Microwave Heating / Chandra, Usha (ur.).
Rijeka, Croatia: InTech, Open Access Publisher, 2011. str. 41-62


Naslov
Role of microwave heating in antigen retrieval in cryosections of formalin-fixed tissues

Autori
Brzica, Hrvoje ; Breljak, Davorka ; Vrhovac, Ivana ; Sabolić, Ivan

Vrsta, podvrsta i kategorija rada
Poglavlja u knjigama, stručni

Knjiga
Microwave Heating

Urednik/ci
Chandra, Usha

Izdavač
InTech, Open Access Publisher

Grad
Rijeka, Croatia

Godina
2011

Raspon stranica
41-62

ISBN
978-953-307-573-0

Ključne riječi
Antigen retrieval, formalin fixation, immunocytochemistry, microwave, paraffin sections, cryostat sections

Sažetak
Immunocytochemistry is a common method for localizing proteins in the mammalian tissues and cultured cells. Samples to be used in immunocytochemical studies are often fixed and preserved with 2%-10% p-formaldehyde (PFA, formalin), frozen or embedded in paraffin, and sectioned. Fixation with PFA denatures the proteins while preserving antigenicity, but it can ‘’mask’’ the antibody binding sites (epitopes), thus diminishing the availability of epitopes and fidelity of immunocytochemical findings. The hidden epitopes can be recovered by using various protocols of antigen retrieval before applying antibodies. These protocols are normally used with sections of the paraffin-embedded tissues, and include heating at high temperatures in defined buffers, treatment with various alcohols, detergents, metals or high pressure, or various combinations of these procedures. Here we report on using similar antigen retrieval protocols on cryosections of the PFA-fixed rat kidney and liver tissues in order to reveal the optimal immunostaining conditions for several representative proteins that reside in specific cellular compartments. The immunostaining was studied by the method of indirect immunofluorescence, where specific epitopes were first labeled with the relevant primary antibody, followed by labeling with the fluorescent molecule-conjugated secondary antibody, and fluorescence microscopy. Our data illustrate: a) that harsh antigen retrieval protocols, previously used with sections of the PFA-fixed, paraffin-embedded tissues, can be efficiently used with cryosections of the PFA-fixed tissues, b) the necessity of testing various antigen revealing protocols for each set of antigen-antibody in order to determine optimal conditions for immunocytochemical presentation, and c) that in most cases the protocols that include microwave heating give the best unmasking effects and staining intensity. Being the strongest unmasking method for different epitopes, microwave heating also generates conditions for significant reduction of the amount of primary antibodies.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti



POVEZANOST RADA


Projekt / tema
022-0222148-2146 - Bubrežni prijenosnici u sisavaca; spolne razlike i učinci toksičnih metala (Ivan Sabolić, )

Ustanove
Institut za medicinska istraživanja i medicinu rada, Zagreb