Mouse anti human cd68 clone pg m1

To investigate distributions of CRegs, an anti-human CD55 mouse monoclonal antibody (Ab) (clone 1C6), kindly donated by Dr. T. Fujita (Fukushima Medecal University, Fukushima, Japan), and an anti-human CD59 mouse monoclonal Ab (clone 1F5), were used as previously reported [18 (link),29 (link),30 (link)]. Monoclonal Ab (mAb) mouse anti-human CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA). As an isotype control immunoglobulin (Ig)G1, mouse anti-rat CD46 (mAb MM.1; Hycult Biotech, Uden, The Netherlands), which is expressed only in male rat genital tissues, was used for immunohistochemical analyses [31 (link)]. Mouse anti-human cytokeratin (clone C-04) and rabbit anti-CD3 (cross-reacted with human) (clone SP7) were purchased from abcam (Kenbridge, UK), and mouse anti-human CD68 (clone PG-M1) was purchased from Dako (Glostrup, Denmark). The mAb anti-activated C3 (aC3, clone bH6; Hycult Biotech) detected depositions of C3b, iC3b, and C3c and the house-made polyclonal Ab (pcAb) anti-human C9, kindly gifted by B. Paul Morgan (Cardiff University, Cardiff, UK), was used to detect the deposition of C5b-9.

As primary antibodies, mouse anti-human CD68 (clone PGM-1, DAKO, Glostrup, Denmark, dilution 1 : 50), mouse anti-human CD163 (clone 5C6, BMA Biomedicals, Augst, Switzerland, dilution 1 : 200), and anti-human CD163 (clone EDhu1, Serotec, Cambridge, UK, dilution 1 : 200) antibodies were used. As negative controls for each antibody, normal mouse IgG1 (Dako) and normal rabbit serum (Vector Labs, Burlingame, CA, USA) were used. The detailed protocol used for immunohistochemistry analysis has been previously described [25 (link)].

YKL-40 and CD68 expression in sputum cytospins was demonstrated using immunofluorescence. Cytospins were fixed in 4 % formaldehyde in PBS for 30 min, followed by antigen retrieval in citrate solution pH6.0 (DAKO, Glostrup, Denmark) for 30 min and cooled on ice. The primary antibodies goat-anti-human YKL-40 (R&D systems, dilution 1:25) and mouse-anti-human CD68 (clone PG-M1, DAKO, dilution 1:50), diluted in PBS/1 % BSA (w/v) were incubated together overnight. Alexa Fluor568 donkey-anti-goat and Alexa Fluor 488 donkey-anti-mouse (Invitrogen, Eugene, Oregon, USA, dilution of both 1:200) were incubated in a dark environment for 30 min. The cytospins were covered with Vectashield with DAPI (Vector Laboratories, Inc. Burlingame, CA, USA). Photographs were taken with a confocal microscope.