Alexafluor 555 goat anti mouse igg h l

AGEs were produced at our laboratory using BSA from Sigma (St. Louis, MO) as substrate proteins. The antibodies against AGEs were raised according the method of Morioka et al. [10 (link)] as mentioned below. The following reagents were purchased from commercial sources: RPMI-1640 medium (Sigma); penicillin-streptomycin solution (Sigma); 200 mM L-glutamine (Invitrogen, Carlsbad, CA); fetal bovine serum 16000 (Invitrogen); PBS, TBS and formaldehyde solution (Sigma); 10 mM Tris-buffered saline and formaldehyde solution (Katayama Chemical Industries, Osaka, Japan); and Triton X-100 (Nacalai Tesque, Kyoto, Japan). Normal goat serum, normal rabbit IgG, AlexaFluor 488-labeled goat anti-rabbit IgG (H+L), and AlexaFluor 555 goat anti-mouse IgG (H+L) were obtained from Abcam (Cambridge, UK); rabbit-caspase-3 antibody from Cell Signaling (Danvers, MA); anti-NF-κB p65 antibody (Rb pAb; 16502) from Abcam; antiphosphatidylserine antibody from Upstate Biotechnology (Lake Placid, NY); goat anti-rabbit IgG (HRP) and DAPI from Life Technology (Carlsbad, CA); and West Dura extended duration substrate, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), and nuclear and cytoplasmic extraction (NE-PER) reagents from Thermo Scientific (Waltham, MA).

Epitopes were retrieved from deparaffinized sections using a heat-induced method. As described before¹⁹ , sections were alternatively bathed in boiling citrate buffer (10 mM citric acid monohydrate, pH 6.0) and Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween-20, pH 9.0). Each bathing step was repeated five times for 2 min each. After permeabilization with Triton™ X-100 (MilliporeSigma; Cat# X100) and washing with 1x PBS, sections were blocked first for endogenous biotin with Endogenous Biotin-Blocking Kit (Thermo Fisher Scientific; Cat# E21390) according to the manufacturer’s instructions and then with 2.5% goat serum in 10 mM HEPES at RT for 1 h. Sections were incubated with primary antibody anti-EPX (clone AHE-1; mouse; 1:200; Abcam; Cat# ab190715) and anti-CD68 (clone FA-11; biotin; 1:100; GeneTex; Cat# GTX43914) in 2.5% goat serum in 10 mM HEPES overnight at 4 °C. After washing in 1x PBS, sections were incubated with the secondary antibodies AlexaFluor®555 goat anti-mouse IgG H&L (1:200; Abcam; Cat# ab150114) and AlexaFluor®488 Streptavidin (1:50; BioLegend; Cat# 405235) in 10 mM HEPES at RT for 3 h. After washing with 1x PBS, sections were mounted with Fluoroshield^TM with DAPI (MilliporeSigma; Cat# F6057) and covered with coverslips. Images were acquired with the All-in-One Fluorescence Microscope BZ-X710 (KEYENCE).