Fluoromount g mounting solution with dapi

Following fixation, gels were dissected into 2mm × 2mm pieces and placed into a 96 well plate. Gentle agitation was provided during staining via a platform shaker at 23°C. Gel pieces were washed in PBS for 30 minutes. They were then permeated with 0.1% TritonX-100 in PBS (ThermoFisher, Cat:85111) for 30 minutes. Gels were then blocked with PowerBlock (BioGenex, Cat: HK085-GP) for 1 hour. Following blocking, primary antibody was made up in a 1:1 solution of 0.1% TritonX-100 and PowerBlock. Primary staining lasted 1 hour, followed by two 15-minute washes with 0.025% Tween 20 in PBS (Sigma, Cat: P1379) and a single 15-minute wash in PBS. Secondary antibody containing Alex Fluor 488, 594, or 647 against the primary antibody was added to a 1:1 solution of 0.1% TritonX-100 and PowerBlock. Secondary antibody staining lasted 45 minutes, followed by two 30-minute washes in 0.025% Tween 20 in PBS (Sigma, Cat: P1379) and a single 30-minute wash in PBS. Stained gel pieces were mounted on a slide with Fluoromount-G mounting solution with DAPI (ThermoFisher Cat:00495952). Fluorescent images were taken with an LSM880 inverted confocal microscope.
The following primary antibodies were used for staining gels: CD45 (Abcam, Cat: ab8216), CD163 (Cat: PA5–78961), and COL1 (Cell Signaling, Cat: 72026S).

Immunohistochemistry (IHC) was conducted on 8 μm thickness sectioned paraffin slides. Following incubation for one hour at 50 °C, slides were washed twice in Tween 20 (Sigma-Aldrich^™, St. Louis, MO) followed by one wash in PBS. Slides were then blocked for 1 hour with Power Block (Biogenex^™, Fremont, CA) prior to addition of the following primary antibodies: CD45 (Abcam, Cat: ab8216), CD163 (Cat: PA5–78961), CD93 (Cat: PA5–52930), CAV1 (Cat: ab214448), CD40 (Cat: 2246639), COL1 (Cell Signaling, Cat: 72026S). Slides were then incubated for 1 hour with Alexa Fluor 488, 594, or 647-conjugated anti-rabbit, anti-rat, or anti-mouse antibodies (Invitrogen, Waltham, MA). Finally, slides were mounted in Fluoromount-G mounting solution with DAPI (ThermoFisher Scientific^™, Waltham, MA). Images were acquired with a LSM880 inverted confocal, Airyscan, AiryscanFAST, GaAsP detector upright confocal microscope.