Alexa fluor 568 anti rabbit antibody

Transfected cells were seeded 24 hr prior to fixing with 4% formaldehyde in PBS in thin-bottomed 384-well plates. The fixed cells were then washed three times in PBS and permeabilized with 0.25% Triton-X-100 in PBS for 5 min at room temperature (RT). After washing with PBS, 5% bovine serum albumin (BSA, Sigma) in PBS was used for 45 min at RT for blocking. The cells were then washed with PBS and incubated with a 1:100 dilution of the anti-MLH1 antibody (Santa Cruz Biotechnology, Product no.: sc-11442) in 1% BSA in PBS for 1 hr at RT. The cells were washed with PBS and incubated with a 1:1000 dilution Alexa Fluor 568 anti-rabbit antibody (Invitrogen) in 1% BSA in PBS for 1 hr at RT. After additional washing with PBS, the DNA was stained with Höchst 33342 (Sigma) for 10 min. Microscopy was performed using an InCell2200 microscope (GE Healthcare). The filters were Höchst (ex 390 nm, em 432 nm) and TexasRed (ex 575 nm, em 620 nm). The InCell Developer Toolbox (GE Healthcare) was used for image analysis. To determine the abundance of the MLH1 variants, the total intensity of the red channel in each cell was measured after excluding the non-transfected cells.

The “EML” method was used to subtype CICs as previously reported (9 (link)). In brief, samples were first stained with antibody against CD45 (mouse mAb from Boster, BM0091) at a dilution of 1:400 by Opal Multiplex tissue staining kit (Perkin Elmer, NEL791001KT) according to the standard protocol provided, and CD45 molecules were eventually labeled with Cyanine 5 fluorophore. Slides were then incubated with mixed antibodies against E-cadherin (mouse mAb from BD Biosciences, 610181) and CD68 (rabbit pAb from Proteintech, 25747–1-AP), followed by secondary antibodies of Alexa Fluor 568 anti-rabbit antibody (Invitrogen, A11036) and Alexa Fluor 488 anti-mouse antibody (Invitrogen, A11029). All slides were counterstained with DAPI to show nuclei and mounted with Antifade reagent (Invitrogen, Carlsbad, CA) and cover slips.
Multispectral images were taken with TMA modules of Vectra® Automated Imaging System (Perkin Elmer) by a 20× objective lens. Nuance system (Perkin Elmer) was used to build libraries of each spectrum (DAPI, FITC, TRITC, and Cy5) and unmix multispectral images with high contrast and accuracy. InForm automated image analysis software (Perkin Elmer) was used for batch analysis of multispectral images based on specified algorithms.