Prolong glass mounting reagent

Cells and tissues were blocked with IF blocking solution for 2–3 h at RT and primary antibodies (diluted in blocking buffer) added and incubated O/N at 4 °C. After 3X wash in PBS-T, fluorescent dye-conjugated secondary antibodies (1:500, Alexa Fluor antibodies from ThermoFisher) were added and incubated for 3 h at RT. Secondary was washed with PBS-T three times and tissues incubated in DAPI (1:1000, ThermoFisher) for 5 min and coverslips mounted with Prolong Gold Antifade Mountant (ThermoFisher P36930). Cells were washed and coverslips mounted with Prolong Glass Mounting Reagent (Thermo Fisher P36981) which contains DAPI.
For BrdU detection, antigen retrieval was performed using HCl. Slides were thawed and rinsed in PBS for 5 min at RT. Samples were incubated in fresh 2 N HCl for 1 hr or 30 min at RT. Samples were rinsed through multiple PBS washes to remove all traces of HCl. For combination IF with SOX2 or YBX1, antibodies were tested for affinity post-HCl in a separate experiment and combined with BrdU for experiments. Antibodies were used as follows: Sox2: diluted 1:200. Ybx1: rabbit/mouse diluted 1:100. BrdU: diluted 1:100. Foxg1: diluted 1:50. Gbx2: diluted 1:100. GFAP: diluted rabbit-1:200, mouse-1:250. Tuj1: diluted 1:100. Images were acquired with Zeiss LSM780 or Keyence BZ-X and image modifications performed with Fiji or Adobe Photoshop.

Cells were seeded on glass coverslips and treated with 40 µM of GlcNAz (Thermo Fisher Cat# PI88905) for 48 h. After three washes with cold DPBS (Gibco Cat# A1285801), cells were Cu-click conjugated with AlexaFluor-488-alkyne (Click Chemistry Tools Cat# 12771) and fixed as previously reported (⁸³ ). Cells were mounted onto glass slides with ProLong glass mounting reagent (Thermo Fisher Cat# P36980). Images were acquired at 60× magnification on the Olympus FV3000RS confocal microscope.
The glycocalyx quantification was determined by defining a region of interest around individual cells and then measuring the intensity of the staining in each region using ImageJ software. Intensity plot of the cell membrane was generated and the width at half maximum intensity was considered as glycocalyx thickness.