Lamp1

Cells were cultured in chamber slides (SPL Life Sciences), fixed with 4% PFA, permeabilized with 0.25% Triton X100, blocked in PBS containing 2% bovine serum albumin (Bio-Rad) and applied with primary antibodies to TrkA (LSBio), Lamp1 (GeneTex). Alexa Fluor antibodies (Life Technologies) were used as secondary antibodies. Isotype control antibodies were used as controls. Cell nuclei were visualized with DAPI (Santa Cruz Biotechnology). Images were captured using a Nexcope NE950 fluorescent microscope (Ningbo Yongxin Optics Co., United States).

PC12 cells were centrifuged at 1000× g for 5 min, and the supernatant was removed. Cell pellet was fixed in 2.0% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PBS, pH 7.4 for 90 min at 4 °C. U87MG cells, after removing the culture medium, were fixed with the same fixing solution (for 90 min at 4 °C), gently scraped from the plate, and centrifuged at 10,000× g rpm for 10 min to obtain the cell pellet.
This fixing solution contained a concentration of aldehyde, which minimally covers antigen epitopes while fairly preserving tissue architecture. After washing, specimens of both cell lines were post-fixed in 1% OsO₄ for 1 h at 4 °C; they were dehydrated in ethanol and finally embedded in epoxy resin.
Plain TEM was implemented by a post-embedding immunocytochemistry procedure for antibodies against LC3 (Abcam, Cambridge, UK) and LAMP1 (Gene Tex, Irvine, CA, USA) used as markers of autophagy vacuoles (autophagosomes and autophagolysosomes, respectively), according to the manuscript “Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (4th Edition)” [87 (link)].
At the end of plain TEM or immunocytochemistry procedure, ultrathin sections were stained with uranyl acetate and lead citrate, and they were finally examined using a JEOL JEM-100SX transmission electron microscope (JEOL, Tokyo, Japan).