Prolong gold antifade reagent with dapi

Cells were grown on coverslips, fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X100 for 10 mins, washed 3 times with PBS and incubated with primary antibody overnight at 4°C. The cells were washed 3 times with PBS and incubated with secondary antibody for 1 hour at room temperature. After washing, coverslips were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). Paraffin-embedded tissue sections were cleared with histoclear (National Diagnostics) and graded alcohol using standard techniques. Antigen retrieval was performed using 7mM citrate buffer, pH 6.0 under pressure. Sections were incubated with primary antibody overnight at 4°C and with secondary antibody for 1 hour at room temperature. Coverslips were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). All images were obtained using a Zeiss 780 confocal microscope and Zeiss LSM 880, and settings were adjusted to allow for detection of fine membrane structure. Primary antibodies were against: LAMP2 (ab13524) from Abcam (Cambridge, MA); PMCA2 (PA1-915) from Thermo Scientific (Waltham, MA); cathepsin B (PA5-17007) and TFEB (PA5-96632) from Invitrogen (Grand Island, NY); Phospho-STAT3 (9145), p21 (2947), p-Rb (8516) and NFAT (5861) from cell signaling (Danvers, MA).

Mouse front limbs (CD1 strain) were collected as fresh post mortem samples. Stages E12, E15, and E18 were examined. Histological sections were deparaffinized in xylene and rehydrated in a gradient series of ethanol. Sections were pre-treated in citrate buffer (10 min/98 °C) for antigen retrieval and then incubated with Caspase-1 p20 (Cleaved Asp296) Antibody (PA5-99390, Thermo Fisher Scientific) overnight. The primary antibody was followed by incubation with secondary anti-rabbit antibody Alexa Fluor^® 488 (Thermo Fischer Scientific, Waltham, MA, USA) for 40 min at RT. Nuclei were detected by ProLong^® Gold Antifade reagent with DAPI (Thermo Fischer Scientific).
For immunocytofluorescence, micromass cultures were grown on culture glass and fixed by 4% paraformaldehyde. Primary antibodies for Caspase-1 p20 (PA5-99390, Thermo Fisher Scientific), Cd36 (PA1-16813, Thermo Fisher Scientific), Pparg (2443, Cell Signaling Technology, Danvers, MA, USA), and Rankl (PA5-110268, Thermo Fisher Scientific) were diluted in the range 1:50–1:200 and were applied overnight/4 °C. Alexa Fluor^® 488 or 568 (A11034, A10037, Thermo Fischer Scientific) was diluted at 1:200 and then applied for 40 min/RT. The cytoskeleton was visualized by ActinGreenTM 488 ReadyProbesTM Reagent (Thermo Fischer Scientific), and nuclei were detected by ProLong^® Gold Antifade reagent with DAPI (Thermo Fischer Scientific).

Cell lines were cultured on poly-L-lysine-coated glass coverslips in 6-well plates with RPMI media supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin for 24 hours. For internalization studies, cells were treated with 200 nM LysoTracker (Invitrogen, Waltham, MA) for 2 hours prior to incubation with 1 μM VGT-309. Coverslips were removed from culture at 60 minutes following VGT-309 treatment. For cathepsin inhibition experiments, cells underwent a 30-minute pretreatment with 100 μM JPM-OEt (MedChemExpress, Monmouth, NJ), a pan-cathepsin inhibitor. Pretreated and non-pretreated cells were then incubated with 1 μM VGT-309 for 1 hour. Following dye administration, coverslips were mounted on glass slides with ProLong Gold Antifade reagent with DAPI (Fisher Scientific, Waltham, MA) and imaged on a Leica DM6 B fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Based on the protocol described previously^{8 (link)}. 4–6-week-old C57BL/6J mice dissociated DRG neurons were plated onto the poly-D-lysine (100 µg/mL), laminin (10 µg/mL) and the aggrecan (50 µg/mL) (P-6407, L-2020, A-1960, Sigma) coated coverslips in the 24-well culture plates and cultured in Neurobasal medium (1088802; Thermo Fisher Scientific) with B27 supplement containing penicillin, streptomycin, 1 mM l-glutamine, 50 ng/mL NGF, 2 ng/mL GDNF, and 10 mM AraC at 37°. For drug treatment, DRG neurons were cultured for 72 h in the presence of the drug phentolamine at 1, 3, 5, 8, 10, 12, 20 μm concentrations. The cells were post-fixed 4% paraformaldehyde (PFA) followed by phosphate buffer saline (PBS) washing and immunostained with anti-mouse b-III-tubulin (1:1000; 801201, BioLegend). Coverslips were then inverted and mounted on the glass-slides using Prolong Gold antifade reagent with DAPI (P36935, Fisher Scientific). Images were captured covering the entire coverslip area at × 20 magnification using an Olympus Fluorescent Microscope. Total neurite length was quantified with an ImageJ plug-in, NeurphologyJ^{38 (link)} (RRID: SCR_003070). Neurphology J operates on the entire image and quantifies the neurite length in pixels. Data were obtained from at least 4 separate experiments repeated in duplicates.

HEL cells transfected with EGFP-tagged constructs were stimulated with 800 nM PMA for 5 min and incubated with immobilized fibrinogen in duplicate, for 1 h or 2 h at 37 °C in serum-free medium. After extensive washing with PBS, the adherent cells were fixed with 4% PFA and stained antivinculin antibody (hVIN-1 clone, Sigma-Aldrich, St. Louis, MO), followed by secondary antimouse goat F(ab) fragment antibody Alexa 568-conjugated (Thermo Fisher Scientific) and with Alexa 647-phalloidin (Thermo Fisher Scientific). The cells were visualized with 40x and 63X objectives using a Leica TCS-NT laser scanning confocal microscope and the cell area was measured for 150 cells with ImageJ software.
HeLa cells after CRISPR or cell sorting were seeded to fibronectin-coated coverslips (10 µg/cm²) for 2 h spreading at 37 °C. Adherent cells were fixed with 4% PFA and stained with anti-vinculin antibody (Sigma) or anti-GFP antibody (Abcam) and anti-ILK antibody (abcam) followed by goat anti-mouse antibody Alexa 488-conjugated (abcam) or goat anti-chicken antibody Alexa 488-conjugated (Abcam) and goat anti-rabbit Alexa 568-conjugated (abcam). Coverslips were then mounted with Prolong Gold Antifade Reagent with DAPI (Fisher) overnight and visualized with Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).