Streptavidin biotin blocking

Organoids were fixed with 4% PFA (Thermo scientific) for 1 h. For whole mounted samples, organoids were blocked using a blocking buffer supplemented with streptavidin/biotin blocking (Vector lab). Tissue was incubated with 1st and 2nd antibodies (Supplementary Table S1) overnight at 4°C and for 2 h at RT, respectively. To enhance the staining, sample clearing was performed by dehydrating the organoid samples in consecutive EtOH treatments at 50, 75, and 100%. To generate cryosections, organoids were soaked in 30% sucrose overnight, and subsequently embedded in OCT compound (Fischer Healthcare). Samples were frozen in liquid nitrogen prior to cryostat sectioning. Tissue sections were placed on glass slides and stored at −20°C. For immunostaining, tissue sections were blocked using blocking buffer supplemented with streptavidin/biotin blocking (Vector lab) and then incubated for 45 min with 1st and then 2nd antibodies (Supplementary Table S1) at RT. Samples prepared using both methods were mounted and observed under the confocal microscope.

Long bones were fixed in 4%PFA, decalcified in 0.5 M EDTA, embedded in gelatin and frozen at −80 °C [27] (link). Immunofluorescence was performed on 30 µm thick cryosections (three non-consecutive levels) for each bone. Tissue sections were permeabilized for 30 min in 0.3% Triton X-100, where appropriate Streptavidin/Biotin blocking (Vector Labs) was performed. Cryosections were incubated for 1 h with primary antibody against murine Endomucin (2 µg/mL, Santa Cruz Biotechnology), Osteopontin (R&D Systems) or human CD29 and CD59 (10 µg/mL, BioLegend). Tissue sections were washed with PBS and incubated with either a fluorescently-conjugated (5 µg/mL) or biotinylated secondary (7.5 µg/mL) antibody followed by fluorescently-conjugated streptavidin. Tissue sections were washed with PBS and incubated with either a fluorescently-conjugated or biotinylated secondary antibody followed by fluorescently-conjugated streptavidin. All incubations were carried out at ambient temperature and ProLong Gold Antifade reagent (Thermo Fisher) to mount the glass coverslips. Images of the immunofluorescence staining were captured with Nikon A1 (Nikon Instruments Europe) or Zeiss LSM 880 AiryScan (Carl Zeiss Microscopy GmBH) confocal systems.