Wga af647

To specifically label afferents from the hind paw skin, 10 μl Alexa Fluor 647-conjugated wheat germ agglutinin (WGA-AF647, Thermo Fisher Scientific, Invitrogen, Eugene, Oregon, USA; 0.8% in sterile PBS) was injected intraplantar into both hind paws. The injections were performed 7 days prior to imaging. Initial experiments showed no edematous or hyperalgesic response to the retrograde label.

The expression level of the RBC membrane proteins was measured by a method developed by our research group (for details, see refs [10 (link),23 (link)]). The RBC membranes were fixed with 1% formaldehyde solution and the resulting RBC membranes (ghosts) were labeled by Alexa Fluor 647 (AF647) conjugated wheat germ agglutinin (WGA-AF647, Thermo Fischer Scientific, Waltham, MA, USA, W32466). Then the samples were labeled with GLUT1 rabbit monoclonal antibody (Abcam, Cambridge, UK, ab115730), reacting with an intracellular epitope, followed by a secondary (Alexa Fluor 488-labeled goat anti-rabbit (H + L) antibody, Thermo Fischer Scientific, Waltham, MA, USA, A-11008). RBC ghosts were analyzed for antibody staining by Attune NxT acoustic flow cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) with excitation wavelengths: 488 nm, emission filters: 530/30 nm for AF488 and excitation wavelengths: 637 nm, emission filters: 670/14, for AF647. The primary and secondary antibodies were titrated to provide maximum labeling.

A Dextran uptake assay in combination with ryanodine receptor (RyR) immunofluorescence was performed to assess viability of the tissue slices according to a published method (Pfeuffer et al., 2023 (link)). Briefly, dextran (3 kDa) conjugated to FITC (Thermo Fisher, D3306) at a concentration of 2 mg/mL was incubated for 10 min at 37°C and 5% CO₂ under continuous rocking on the MyoDish culture system platform. The slices were immediately fixed with 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min and then washed three times with PBS for 5 min each. Subsequently, slices were incubated with primary antibody against cardiac RyR (IgG1, mouse, C3-33, Thermo Fisher, Braunschweig, Germany) 1:200 in blocking solution (BS: 5% NGS, 5% BSA, 0.25% Triton-X in PBS) for 4 h at RT or overnight at 4°C, washed three times with PBS for 5 min and incubated with the secondary antibody goat anti-mouse IgG1 AF-555 (Thermo Fisher A-21127) 1:400 in BS for 3 h at RT. After washing, the slices were incubated with wheat germ agglutinin (WGA-AF-647 Thermo Fisher W32466) 40 μg/mL and DAPI (3665, Roth, Karlsruhe, Germany) 1.67 μg/mL in PBS for 3 h. The slices were mounted with Fluoromount G (00-4958–02, Thermo Fisher; F4680 Sigma-Aldrich, Darmstadt, Germany) on a glass microscope slide, covered with a coverslip and equilibrated for 3–7 days at 40%–45% humidity.

CD8 T cells were preactivated with T cell activator beads CD3/CD28. Before CD137 stimulation through CD137 (4-1BBL)–Fc–coated plates, a certain fraction of cells was preincubated with BV6 2 hours at 37°C to ensure cIAP degradation. After 15 min of CD137 stimulation, cells were fixed with 4% PFA and stained with wheat germ agglutinin (WGA) AF647 (Life Technologies), anti-CD137–AF488 (BioLegend), and HOETSCH. Images were collected using an LSM 800 confocal microscope (Zeiss). Images were analyzed using ImageJ, generating manual region of interest (ROI) in the cytoplasm of each CD8 T cell and quantifying the mean fluorescence intensity of CD137 signal in each cell cytoplasm.

Primary antibodies included the following: EEA1 (Santa Cruz Biotechnology, sc-6415), Rab7 (Abcam, ab 137029), Lamp1 (BD Pharmingen, 1D4B), GFP (Life Technologies, A11122). Secondary antibodies included the following: donkey anti-goat–AF555 (Invitrogen, A21432), goat anti-rat–AF488 (Invitrogen A11006), chicken anti-rabbit–AF488 (Invitrogen, A21441).
Other reagents included the following: AF594/Fab (Jackson ImmunoResearch Laboratories Inc., 309-586-003), A488 phalloidin (Life Technologies, A12379), AF555 phalloidin (Life Technologies, A34055), DQ Green BSA (Life Technologies, D12050), Tf-AF647 (Life Technologies, T23366), dextran-AF488 (Life Technologies, ID7178), WGA-AF647 (Life Technologies, W32466).