Alexa fluor 594 conjugated anti rabbit igg antibody

The levels of Microtubule-associated protein 1 light chain 3 (LC3)-II and lysosomal associated membrane protein (LAMP)-1 were visualized through confocal microscopy following immunofluorescent staining. The cells were exposed to primary antibodies targeting LC3-II (1:400, Medical and Biological Laboratory Co., Ltd., Woods Hole, MA, USA) and LAMP-1 (1:400, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBS, the sections were incubated with a 1:500 dilution of Alexa fluor 488-conjugated anti-mouse IgG antibody (Cell signaling) or Alexa fluor 594-conjugated anti-rabbit IgG antibody (1:500 dilution; Cell signaling) for 1 h in the dark at room temperature. Subsequently, the nuclei were stained with 4′,6-diamidino-2-Phenylindole (DAPI) for 5 min, and then the samples were mounted. For the negative control, the primary antibody was omitted, and the sections were incubated with the corresponding secondary antibodies and the detection solutions. A confocal microscope (LSM 700; Zeiss, Jena, Germany) was used to capture fluorescence images Immunofluorescent staining evaluation involved the calculation of mean fluorescence intensity (MFI) using the ImageJ software (https://imagej.nih.gov/ij/download.html, accessed on 1 June 2022) in a blinded manner [6 (link),22 (link),23 (link)].

Immunofluorescence was performed as described previously 38 (link). In brief, cells were transfected with miR-204-5p mimics or inhibitors and then fixed with 4% para-formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 1% BSA in PBS, and subsequently incubated with primary antibodies. The primary antibodies were as listed in Table S4. The cells were then incubated with Alexa Fluor 488-conjugated anti-rabbit IgG antibody (#4412, Cell Signaling Technology, Danvers, MA, USA) and Alexa Fluor 594-conjugated anti-rabbit IgG antibody (#8889, Cell Signaling Technology) at room temperature for one hours in the dark. The cells then washed with PBS and counterstained with DAPI (Invitrogen) for 5 min following the manufacturer's protocol. Images were obtained with a laser scanning microscope (Carl Zeiss, Jena, Germany).