Alexa fluor 555 conjugated goat anti mouse igg h l secondary antibody

Frozen lung tissue samples of rats were sectioned at 7 µm intervals and fixed with 4% paraformaldehyde. For the detection of MPO-positive cells (neutrophils, monocytes), sections were permeabilized with 0.2% Triton X-100 and incubated with 5% normal goat serum + 0.5% BSA + 0.2% TritonX-100 in PBS in order to block unspecific binding. Sections were further incubated with anti-MPO antibody (1:100, Abcam) for 1 h followed by additional incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000, Cell Signaling Technology) overnight at 4 °C.
For the detection of leukocytes, a mouse monoclonal anti-rat CD45 antibody was used (clone OX-1, 1:20, BD Pharmingen). Then, slides were further incubated with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:1000, Thermo Fisher) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenyl indole (DAPI) and slides were mounted in fluorescence mounting medium (Dako). Tissue sections were examined using an inverted microscope (Eclipse Ti-U 100, Nikon). Signal intensity of CD45 immunolabeling in ten random fields was quantified as mean value and averaged using the Image J analysis software.

ARPE-19 cells transiently transfected with green fluorescent protein (GFP)-LC3 were treated with 100 μM CQ with or without 2 μM PPD, fixed with 4% (v/v) paraformaldehyde (PFA), and subsequently permeabilized with PBS containing 0.2% (v/v) Triton X-100 and 1% bovine serum albumin (BSA) at room temperature. After washing with PBS, the cells were blocked for 1 h and incubated with LAMP-2 antibody at 4°C overnight. Then, the cells were incubated with an Alexa Fluor 555-conjugated goat anti-mouse IgG (H+L) secondary antibody (#A28180, Thermo Fisher Scientific) for 1 h at room temperature. The fluorescence intensity and protein localization were analyzed using a Zeiss LSM710 laser confocal microscope (Carl Zeiss). Quantification of the number and size of GFP-LC3-positive cells and colocalization in the acquired images was performed using ImageJ software (National Institutes for Health [NIH], Bethesda, MD, USA). Mean autophagosome counts from 100 cells per treatment were obtained from three separate experiments.