Alexa fluor 647 conjugated secondary antibody

HCT116 cells exponentially growing on sterilized cover glass slides (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) were treated with gefitinib, with or without NDAT or PI3K inhibitor (LY294002) (Selleck Chemicals). The cells were immediately fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Fluorescein-labeled Sambucus nigra lectin (FITC-SNA, Vector Laboratories, Burlingame, CA, USA) that preferentially binds to α2,6-linked sialic acid structure was used to investigate the product of ST6Gal1 after these treatments. Cells on the slides were incubated with anti-EGFR antibody (GeneTex) overnight at 4 °C and then incubated with Alexa Fluor®-647-conjugated secondary antibody (Abcam, Cambridge, UK) for 1 h at room temperature (kept in the dark). Cells were then incubated with FITC-SNA (Vector Laboratories) for 15 min at room temperature (kept in the dark). All slides were mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Hayward, CA, USA). The fluorescent signals from EGFR and FITC-SNA were recorded and analyzed with TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems, Wetzlar, Germany). The figures shown are representative of four fields for each experimental condition. Nuclei were defined with DAPI staining.

The glioma cells were seeded on slides and fixed with 4% paraformaldehyde (PFA) for 10 min. Thereafter, the cells were incubated with anti‐β‐catenin (1:200; Abcam Cat#: ab223075; RRID: none; Abcam Inc.) at 4℃ overnight and warm‐incubated with Alexa Fluor® 647‐conjugated secondary antibody (1:500; Abcam Cat#: ab150075; RRID: AB_2752244; Abcam Inc.) for 1 h. The nuclei were stained with Hoechst. The images were captured under an inverted microscope (Ti2‐E, Nikon Instruments Inc., Tokyo, Japan).

Fisetin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), LPS from Escherichia coli O111:B4, ATP disodium salt hydrate, neutral red, and N-phenylthiourea (PTU) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), antibiotic mixture, and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from WelGENE (Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea). Antibodies against ASC (sc-22514), caspase-1 (sc-56036), p50 (sc-8414), p65 (sc-8008), LC3 (sc-376404), p62 (sc-48402), nucleolin (sc-13057), and β-actin (sc-69879) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibodies against NLRP3 (15101S) was purchased from Cell Signaling Technology (Beverly, MA, USA). Thermo Fisher Scientific (Waltham, MA, USA) and GenTex (Zeeland, MI, USA) supplied specific antibodies against IRAK (PA5-20018) and MyD88 (GTX-112987), respectively. Peroxidase labelled anti-mouse immunoglobulins (sc-16102) and anti-rabbit immunoglobulins (KO211708) were purchased from Santa Cruz Biotechnology and KOMA Biotechnology (Seoul, Republic of Korea). Alexa Fluor 647-conjugated secondary antibody was purchased from Abcam (Cambridge, MA, UK). Dako Faramount Aqueous Mounting Media was obtained from Dako (Carpinteria, CA, USA). All other chemicals were purchased from Sigma-Aldrich.

Pancreatic tissue or isolated islets were embedded in Tissue-Tek OCT Compound (VWR, Missouri City, TX, USA), and then, snap-frozen. Frozen sections (10 μm) were cut, placed on Superfrost glass slides, and then, stored at −80°C. For immunostaining, sections were fixed in methanol, followed by overnight incubation with primary antibodies raised against insulin (Santa Cruz Biotechnology, Dallas, TX, USA; 1:200 dilution), collagen IV, VI, fibronectin (Abcam, Cambridge, MA, USA; 1:200 dilution), and CD31 (R&D Systems, Minneapolis, MN, USA; 1:100 dilution). Alexa Fluor 647 conjugated secondary antibodies (Abcam; 1:500 dilution) were used for detection.
For immunofluorescence of paraffin embedded rat islets, blocks were cut into 4 μm sections, deparaffinized, rehydrated, and subjected to antigen retrieval with citrate buffer using a microwave oven on high power. Sections were immunostained with anti-insulin (see above) primary antibody followed by Alexa Fluor 488 conjugated antibody (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution).
Both frozen and paraffin sections were mounted in Fluoroshield mounting media containing DAPI (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using an Olympus IX73 fluorescence microscope or Zeiss LSM 710 confocal microscope.