Nbp1 49955

Cells were grown on coverslips, fixed in −20 °C 100% methanol for 10 min on ice, and permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT). Cells were incubated with blocking buffer (1% BSA, 0.1% Tween 20 in PBS) for 1 h at RT. Coverslips were incubated with primary antibodies for 1 h at RT, washed three times with 1% PBS and once with blocking buffer, and incubated with the appropriate fluorescent secondary antibody for 1 h at RT. The coverslips were then washed three times with 1% PBS and mounted on microscopy slides using mounting reagent with DAPI (Ibidi GmbH, Gräfelfing, Germany). Staining was analyzed using a Leica DMi8 inverted microscope with a 63x oil-immersion objective using the same laser parameters. All microscope images were analyzed with ImageJ software. Primary antibodies used: anti-RAD50 mouse monoclonal antibody (GTX70228, GeneTex), anti-NBS1 rabbit monoclonal antibody (#14956, Cell Signaling Technology), anti-MRE11 mouse monoclonal antibody (sc-135992, Santa Cruz Biotechnology), anti-p62 rabbit polyclonal antibody (NBP1-49955, Novus Biologicals), anti-LC3 rabbit polyclonal antibody (NP100-2220, Novus Biologicals) and anti-LAMP1 mouse monoclonal antibody (sc-20011, Santa Cruz Biotechnology).

All tissues were fixed in formalin-acetic acid-alcohol (FAA) for 24 h at 4°C, subsequently embedded in paraffin, and sectioned to a thickness of 4 μm. HE staining was performed following standard histological procedures [38 (link)]. IF staining was performed as previously described [39 (link)]. The primary antibodies used for the IF were as follows: mouse anti-GRP78 (1:100, sc-376768, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-BCL2 (1:50, ab32124, Abcam, Cambridge, UK), mouse anti-Beclin1 (1:50, NBP1-00085) rabbit anti-p62 (1:200, NBP1-49955, Novus, Littleton, USA) and FOXL2 (1:50, 19672-1-AP, Proteintech, Wuhan, China). Sections were viewed under an Eclipse 80i microscope (Nikon, Tokyo, Japan). The fluorescence images of the slides were visualized using an IX70 fluorescence microscope (OLYMPUS, Tokyo, Japan). The tissue stained without adding primary antibody was considered to be a negative control.