Lightning link conjugation kit

Tissue samples of the quadriceps femoris taken from young and old mice (n = 5 each) were treated with a 20 mL solution of 0.1% collagenase (Catalog Number. 03222364, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 37 °C for 1 h. The digested samples were then filtered through a nylon mesh filter (pluriStrainer 100 µm, pluriSelect, Leipzig, Germany) to obtain cell suspensions. The cells were then treated with the following antibodies: anti-CD45-PE/Cy7 (Clone: 30-F11, Catalog number 103113, BioLegend, CA, USA) and anti-Sca1-APC-Cy7 (Clone: D7, Catalog number 108126, BioLegend) for 1 h at 4 °C. CD45 is a marker for pan-hematopoietic cells and Sca1 is a marker for mature myocytes. After further treatment with a fixation/permeabilization solution (catalog number 420801, BioLegend), the cells were exposed to FITC-conjugated anti-APJ antibody, prepared using an FITC conjugation kit (Lightning-Link conjugation kit, Abcam) and unlabeled anti-APJ antibody (Cat. No. 20341-1-AP, Proteintech, CA, USA) for 30 min at 4 °C. After washing in wash buffer twice, the labeled cells were used for flow cytometry. The procedure involved acquiring 50,000 total events using a BD FACSVerse system (BD Biosciences, San Jose, CA, USA) and analysis of the findings using FlowJo v10.7 (Tree Star, Ashland, OR, USA). Negative gates were set based on the isotype control.

Flow cytometry was performed with antibodies listed in Supplementary Table 2. Dead cells (identified using 7-AAD Viability Staining Solution or Zombie NIR Fixable Viability Kit; both from BioLegend) and cell aggregates (identified on FSC-A versus FSC-H scatter plots) were excluded from all analyses. For intracellular staining, surface-labelled cell suspensions were fixed using eBioscience Foxp3/Transcription Factor Staining Buffer Set or eBioscience IC Fixation Buffer (both from Thermo Fisher). HMOX-1 expressing cells were detected with anti-HMOX-1 antibody coupled to PE using the Lightning-Link conjugation kit (abcam). Data acquisition was performed on an Aria-II SORP, ARIA-Fusion or LSR-Fortessa (BD Biosciences) and analysed using FlowJo software (BD Biosciences). Sorting was performed on an Aria-II SORP or ARIA-Fusion (BD Biosciences).

Organoids were washed once in PBS at room temperature and enzymatically digested using Neuron Isolation Enzyme (Thermo Fisher Scientific, 88285). Cells were washed in FACS buffer (1% bovine serum albumin in PBS) and fixed in 4% PFA. Permeabilisation was achieved using FACS buffer with 0.1% saponin (Thermo Fisher Scientific, A18820.22). Cells were incubated with PAX6 antibodies conjugated to allophycocyanin (Lightning Link Conjugation Kit, Abcam, ab201807) for 30 min. Cells were washed once more in FACS buffer and stained with Hoechst 33342. Flow cytometry was performed on the FACS Canto BD flow cytometer (BD Biosciences). The mean fluorescence intensity of 50,000 cells was measured and statistical analysis was performed as described below.

Two-fold serial dilutions of cyclophilin A or cyclophilin B (both from Sigma-Aldrich) at starting concentrations of 5 µg/mL were absorbed in 200 µL volumes to nitrocellulose using the BioRad (Hercules, CA) slot blot apparatus. After Ponceau stain, the membrane was blocked for 30 min at room temperature in a 4% milk solution of Tris-buffered saline with 0.05% Tween-20 (TBST). The membrane was then incubated overnight at 4°C in 15 mL of TBST/4% milk containing 5 µg of PTS1 (Aviva Systems Biology, San Diego, CA) labeled with horseradish peroxidase via the Lightning Link conjugation kit (Abcam). Chemiluminescence was used to visualize membrane-bound PTS1.

Spleens were fixed in 4% (wt/vol) paraformaldehyde (PFA) for 10 min, saturated overnight in 30% (wt/vol) sucrose at 4 °C, and embedded in Tissue-Tek optimum cutting temperature compound (Sakura) followed by freezing in −80 °C. For detection of Ly6C and BST-1, sections were fixed prior to staining with ice-cold acetone for 10 min. Sections (7 μm) were blocked with 10% (vol/vol) goat serum. Endogenous peroxidase and biotin activities were quenched respectively with 3 % (vol/vol) hydrogen peroxide solution and Endogenous Biotin-Blocking Kit (Thermo Fisher). Antibodies (listed in Supplementary Table 2) were diluted in PBS containing 0.05% (vol/vol) Tween-20 and 2% (vol/vol) goat serum. Primary biotinylated antibodies were visualized with HRP-conjugated streptavidin followed by TSA Plus Cyanine 3 or Cyanine 5 System (Akoya). BST-1 expressing cells were detected with anti-BST-1 antibody coupled to Alexa647 using the Lightning-Link conjugation kit (abcam). Images were acquired with ZEISS LSM 980 confocal microscope and analysed using ZEN software (both from Carl Zeiss MicroImaging).

For covalent labeling of lectins with fluorophores targeting the primary amine groups of lectins, Lightning Link conjugation kits were used following the simple and rapid procedure provided by the manufacturer (Abcam, Cambridge, US). Aleuria Aurantia lectin (AAL, Cat. L-1390-2, Vector Laboratories, Newark, USA) was labeled with APC (Cat. ab201807, Lot.GR3388854-1), recombinant human galectin-1 as a gift of Dr. Éva Monostori, Gal-1, expression and purification steps described in detail in (44 (link)–46 (link)), that was conjugated to PE/Cy7 (Cat. ab102903, Lot. GR3390407-1), recombinant human sialoadhesin, Siglec-1/CD169 protein (Siglec-1, Cat. 5197SL050, Fischer Scientific, Massachusetts, USA) was labeled with PE/Texas Red (Cat. ab269899, Lot. GR3395603-2), and finally galectin-3 (Gal-3, Cat. 450-38, PeproTech, London, UK) was chemically linked to APC/Cy7 dye (Cat. ab102859, Lot. GR3396716-1). Fluorescein-conjugated Sambucus nigra agglutinin was purchased from Vector Laboratories (Cat. FL-1301-2). The list of the selected lectins for cytometry is listed in Supplementary Table 4.