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Naslov:Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry
Avtorji:ID Hira, Vashendriya V. V. (Avtor)
ID Jong, Annique Loncq de (Avtor)
ID Ferro, Klea (Avtor)
ID Khurshed, Mohammed (Avtor)
ID Molenaar, Remco J. (Avtor)
ID Noorden, Cornelis J. F. van (Avtor)
Datoteke:.pdf PDF - Predstavitvena datoteka, prenos (13,71 MB)
MD5: 9CA91EABCFFFFFBC063B3242BBB85421
 
URL URL - Izvorni URL, za dostop obiščite https://doi.org/10.1016/j.acthis.2018.10.011
 
Jezik:Angleški jezik
Tipologija:1.01 - Izvirni znanstveni članek
Organizacija:Logo NIB - Nacionalni inštitut za biologijo
Povzetek:Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.
Ključne besede:immunohistochemistry, chromogens, aminoethyl carbazole, AEC, 3, 3'-diaminobenzidine, DAB, antigen retrieval, tonsil
Status publikacije:Objavljeno
Verzija publikacije:Objavljena publikacija
Datum objave:01.02.2019
Leto izida:2019
Št. strani:str. 125-134
Številčenje:Vol. 121, iss. 2
PID:20.500.12556/DiRROS-19588 Novo okno
UDK:577
ISSN pri članku:0065-1281
DOI:10.1016/j.acthis.2018.10.011 Novo okno
COBISS.SI-ID:5186895 Novo okno
Datum objave v DiRROS:24.07.2024
Število ogledov:396
Število prenosov:272
Metapodatki:XML DC-XML DC-RDF
:
HIRA, Vashendriya V. V., JONG, Annique Loncq de, FERRO, Klea, KHURSHED, Mohammed, MOLENAAR, Remco J. in NOORDEN, Cornelis J. F. van, 2019, Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry. Acta histochemica [na spletu]. 2019. Vol. 121, no. 2, p. 125–134. [Dostopano 6 april 2025]. DOI 10.1016/j.acthis.2018.10.011. Pridobljeno s: https://dirros.openscience.si/IzpisGradiva.php?lang=slv&id=19588
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Gradivo je del revije

Naslov:Acta histochemica
Skrajšan naslov:Acta histochem.
Založnik:=Gustav= Fischer Verlag
ISSN:0065-1281
COBISS.SI-ID:24852224 Novo okno

Gradivo je financirano iz projekta

Financer:Drugi - Drug financer ali več financerjev
Program financ.:Dutch Cancer Society
Številka projekta:UVA 2014-6839 and 2016-10460

Licence

Licenca:CC BY-NC-ND 4.0, Creative Commons Priznanje avtorstva-Nekomercialno-Brez predelav 4.0 Mednarodna
Povezava:http://creativecommons.org/licenses/by-nc-nd/4.0/deed.sl
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