Formalin-fixed, paraffin-embedded sections were immunostained for CD3 (ready-to-use (RTU) rabbit polyclonal anti-human antibodies; Dako, Glostrup, Denmark), CD4, CD8, CD20, CD68, CD31 (RTU mouse monoclonal anti-human antibodies; Dako) and peripheral node addressin (PNAd) clone MECA-79 (1/3000; Biolegend, San Diego, CA, USA). All immunostains were performed on 5-μm-thick paraffin sections using the Dako autostainer. For visualisation, the Envision Flex system (Dako) was used and antigen retrieval was carried out using the Dako pretreatment module at pH=6. For PNAd, an additional incubation of 30 min was necessary with a rabbit anti-rat immunoglobuline (1/75 +10% normal human serum, Dako).
Pretreatment module
Manufactured by Agilent Technologies
Sourced in United States
The Pretreatment module is a laboratory equipment designed to prepare samples for subsequent analysis. It performs essential sample preparation steps, such as dilution, filtration, or extraction, to ensure the sample is in the appropriate state for analysis by other analytical instruments.
Automatically generated - may contain errors
2 protocols using pretreatment module
1
Immunohistochemical Characterization of Tissue
2
Immunohistochemical Quantification of Ki-67 in Menstrual Cycle
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Tissue cores were placed in 10% neutral buffered formalin within 5 minutes of acquisition and delivered to IU Health Pathology for routine paraffin embedding. Sections 3 to 5 microns thick were deparaffinized and hydrated to running water. Antigen retrieval was carried out in the pretreatment module (DAKO, Carpinteria, CA, USA) using low pH target retrieval (DAKO). All staining was performed on the AutoStainer Plus (DAKO). Sections were incubated with 3% H2O2 for 5 minutes and subsequently exposed to the primary antibody, Ki-67 (Mib-1, DAKO), for 20 minutes. Horseradish peroxidase-labeled secondary antibody (EnVision™/HRP) was placed on the tissue for 20 minutes followed by 3,3′-diaminobenzidine (DAB) + chromogen for 10 minutes. Sections were counterstained with EnVision™ Flex Hematoxylin (DAKO). The pathologist (SB) was blinded as to the phase of the menstrual cycle or to the use of hormonal contraception (HC). Each section was classified from grade 1 to 4 where 1 is the lowest number of cells stained/slide and 4 is highest number of cells stained/slide.
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