Alexa fluor 546 goat anti rabbit secondary antibody

Staining for Fos was performed using a general immunofluorescence protocol. Following a series of washes in 0.01 m PBS containing 0.5% Triton X-100, slices were incubated overnight in a PBS blocking solution containing 2% NGS, 0.5% Triton X-100, and 2.5% bovine serum albumin at 4°C. Then, slices were washed in PBS and incubated for 24 h at RT in rabbit polyclonal primary antibody (1:2000; Santa Cruz Biotechnology) in blocking solution. After a series of PBS washes, slices were incubated for 2 h at RT in Alexa Fluor 546 goat anti-rabbit secondary antibody (1:250; Life Technologies). After a series of PBS washes, tissue was floated onto slide glass and coverslipped.

Frozen tumor sections (8 µm thick) were cut axially from two regions for each tumor, one in the center of the tumor and one 1 mm apart. To assess apoptosis, acetone-fixed sections were stained with a rabbit polyclonal anti‐cleaved caspase‐3 (CC3) antibody [AB3623] (1/200, Sigma-Aldrich) and Alexa Fluor 546 goat anti‐rabbit secondary antibody (2/1,000, ThermoFisher). Non‐immune‐specific rabbit IgG in the same concentrations as the anti‐CC3 antibody, was used as a negative isotype control. Staining was visualized under an Eclipse Ni-E Nikon fluorescent microscope using a Nikon DS-Fi3 camera (Nikon Instruments, Surrey, UK). For each tumor (n = 6 per treatment cohort), images were acquired from five randomly selected areas for each of the two tumor sections.
To assess the degree of necrosis, sections were stained with hematoxylin and eosin (H&E) and images were acquired using a bright-field Hamamatsu NanoZoomer 2.0RS digital slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan). Tumor necrotic areas and fluorescent areas for CC3 were defined and analyzed blinded using ImageJ2 software (25 (link)) as previously described (26 (link)). Necrosis was expressed as a percentage of the whole tumor section area, while fluorescent staining as a percentage of the total image area.

Neutrophils (5 × 10⁵) adhered to 0.001% poly-L-lysine-coated slides were stimulated with CSFE-labeled amastigotes (10⁵) for 90 min at 35 °C and 5% CO₂, and fixed in 4% formaldehyde (Sigma, St. Louis, MA, USA). The NETs were stained with rabbit anti-human elastase antibody (1:400 dilution; Calbiochem, La Jolla, CA, USA) diluted in blocking solution (PBS, 3% BSA) and Alexa Fluor 546-goat anti-rabbit secondary antibody (1:300 dilution; Thermo Scientific, Eugene, OR, USA) for 40 min. Slides mounted in ProLong Gold Antifade Mounting media with DAPI (10 μg/mL; Thermo Fischer, Eugene, OR, USA) were imaged in a Zeiss DMi8 confocal microscope (Zeiss, Oberkochen, Germany) and analyzed using an LAS AF imaging program.

Slides of fixed MDM from days 3, 6, 9, and 12dpi were washed twice with PBS 1X and incubated with blocking buffer (1% BSA and 1% goat serum in PBS 1X) for 1 h in a humidity chamber, shaking at RT. MDM were incubated with a rabbit polyclonal antibody raised against the N-terminal region of human CB2R (1:100; Cayman Chemical) in blocking buffer overnight, shaking at 4ºC. The next day, cells were washed five times with Tris-buffered saline (TBS 1X) and incubated with a goat anti-rabbit Alexa Fluor® 546 secondary antibody (1:500 Thermo Fisher Scientific) in a blocking buffer for 1 h in a dark humidity chamber, shaking at RT. Cells were washed five times with TBS 1X and counterstained with VECTASHIELD® Antifade Mounting Media with DAPI (Vector Laboratories). Fluorescence microscopy was performed using a Nikon Eclipse E400, with camera SPOT Insight QE and Fluorescence X-cite Series 120 under an excitation wavelength of 546 nm and 405 nm for CB2R and nuclei, respectively. The magnification was set at 20X. At least 3 different random pictures per condition were acquired. The mean fluorescence intensity (MFI) values of CB2R were acquired using the NIS Elements Analysis software (Nikon).

A suspension of bacteria were incubated with SCPPPQ1 (20 µM final concentration) or the same volume of buffer only (negative control) on coverslips at 37 °C during 20 min. Samples were then fixed 30 min with 4% PFA and 0.1% glutaraldehyde. Coverslips were then rinsed three times with 0.1 M PB before blocking for 1 h with 5% milk in 0.1 M PB. Next, coverslips were incubated for 1 h in 0.5% milk in 0.1 M PB with 1:500 rabbit antibody anti-human SCPPPQ1 (GenScript, Piscataway, NJ, USA) at RT. They were rinsed three times in 0.1 M PB and incubated for 1 h with 1:1000 goat anti-rabbit Alexa Fluor-546 secondary antibody (Thermo Fisher Scientific) at RT. Coverslips were then rinsed three times in 0.1 M PB, incubated 15 min with 1:500 SYTO 9 green fluorescent nucleic acid stain (Thermo Fisher Scientific) and finally mounted with ProLong Gold Antifade Mountant for observation. The samples were examined under the same conditions as describe above. Briefly, Z-stack volumes were acquired using SIM on an Elyra PS1 microscope and reconstructed using the Zen Black edition software version 14.0.18.201 as described above in the fluorescencestudies section.

Transduced U2-OS cells were seeded into six-well plates containing sterile glass coverslips. After two washes with ice-cold PBS, cells were fixed in 4 % (v/v) paraformaldehyde, quenched in 150 mM ammonium chloride, permeabilized with 0.1 % (v/v) Triton X-100 in PBS and blocked for 30 min in 5 % (w/v) FBS in PBS. The cells were stained with rabbit anti-FLAG antibody (Sigma) for 1 h, followed by incubation with goat anti-rabbit Alexa Fluor 546 secondary antibody (Invitrogen). Coverslips were mounted in MOWIOL 4-88 (Calbiochem) containing DAPI. Images were taken on a Zeiss LSM780 confocal microscope using Zen2011 acquisition software. Following vv811-GAAP infection, U2-OS cells were stained using rabbit anti-GAAP polyclonal antiserum (Gubser et al., 2007 (link)) and mouse anti-GM130 antibody (BD Biosciences) to check GAAP expression and localization.