Rabbit anti ccr2

Coronal sections were air-dried, washed with 1X PBS, blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO, USA) in 0.2% triton-X100 for 1 h, incubated with a primary antibody diluted in blocking solution overnight at room temperature (RT), and then washed with 1X PBS six times 10 min each and incubated with appropriate secondary antibody solution (1:250 dilution) for 1 h at RT. Slides were then washed with 1X PBS and then mounted with media-containing DAPI counterstain (SouthernBiotech, Birmingham, AL, USA). Antibodies used are goat anti-EphA4 at 1:100 dilution (R&D systems, Minneapolis, MN, USA), rabbit anti-TMEM119 at 1:100 dilution (Abcam, Waltham, MA, USA), rabbit anti-CCR2 at 1:100 dilution (Abcam, Waltham, MA, USA), rat anti-CD68 at 1:100 dilution (Thermofisher, USA), rabbit anti-Iba1 at 1:200 dilution (FUJIFILM Wako Chemicals U.S.A. Corporation), donkey anti-goat 594 (Thermofisher, USA), donkey anti-rabbit 647 (Thermofisher, USA), donkey anti-rabbit 555 (Thermofisher, USA), and donkey anti-rat 647 (Thermofisher, USA). Image acquisition was performed using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

Images were collected using a Leica DMI 4000B fluorescence microscope. To detect caveolin-1, CCR1, CCR2, and CCR3, cells isolated as described above were cultured overnight in 6-well tissue culture plates (1 × 10⁶ cells per well) on coverslips in RPMI 1640/20 % FCS. Cells were then fixed and permeabilized, labeled with appropriate primary and secondary antibodies, and counterstained with the nuclear stain DAPI.
Immunohistochemistry of human lung tissue sections was performed as described [11 (link)]. Briefly, paraffin sections were stained with primary antibodies, appropriate AlexaFluor647- or AlexaFluor555-conjugated secondary antibodies and the nuclear stain DAPI (Invitrogen, Carlsbad, CA). Images were collected using a Leica DMI 4000B fluorescence microscope. Primary antibodies were: rabbit anti-CCR1 (Thermo Fisher Scientific, Rockford, IL, USA; PA1-21629), rabbit anti-CCR2 (Abcam, Cambridge, MA, USA; ab32144), rabbit anti-CCR3 (Abcam, Cambridge, MA, USA; ab36827), rabbit anti-MCP-1 (Abcam, Cambridge, MA, USA; ab 9669), rabbit anti-MCP-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374002), rabbit anti-pSrc-Tyr416 (Cell Signaling Technology, Inc., Danvers, MA, USA; #2101S), and rabbit anti-pLyn-Tyr507 (Cell Signaling Technology, Inc., Danvers, MA, USA; 04–375).

The following antibodies were used: mouse anti-Pax7 (1:100, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA), mouse anti-MyoD (BD Biosciences #554130), mouse anti-phospho-MyoD (1:1000, Sigma) rabbit anti-Myogenin (1:250, AbCam), rat or rabbit anti-Laminin (1:1000 or 1:1500, Sigma-Aldrich, L0663 or L9393), rabbit anti-skeletal muscle myosin (1:250, Sigma-Aldrich HPA1239), rabbit anti-Ccr2 (1:500, AbCam), rabbit or mouse anti-p38 (1:1000, Cell Signaling), rabbit or mouse anti-phospho-p38 (1:1000, Cell Signaling), rabbit anti-phospho-p38delta/gamma (1:1000, ThermoFisher), rabbit anti-Erk1/2 (1:1000, Cell Signaling), mouse anti-phospho-Erk1/2 (1:1000, Cell Signaling). All antibodies were previously confirmed for specificity in the literature and/or by the manufacturers.