Cryosections (10 μm) were cut from mouse and human samples, mounted on slides (Superfrost Plus; VWR, Leicestershire, UK), and left to dry at RT. Cryosections from unfixed tissue were fixed in acetone (Sigma-Aldrich) for 10 minutes at −20°C prior to further processing. For IBA-1 staining, adjacent sections from unfixed tissue were fixed with 4% (wt/vol) PFA for 10 minutes prior to further processing. Sections from PFA-fixed human tissue required antigen retrieval prior to IHC; sections were heated to 110°C in a solution of 90% glycerol and 10% 0.01 M citrate buffer, pH 6.0 for 10 minutes. Sections were then incubated in blocking buffer (1% [wt/vol] bovine serum albumin, 0.5% [wt/vol] Tween20 in PBS, all reagents from Sigma-Aldrich) for 1 hour at RT, followed by 1 hour at RT in primary antibodies diluted in blocking buffer. Sections were treated in wash buffer (0.5% [wt/vol] Tween20 in PBS) and incubated in secondary antibody diluted in wash buffer for 1 hour at RT. Sections were then treated with wash buffer, counter stained with Hoechst, washed with PBS, mounted in mounting medium (Mowiol 4-88; Merck Millipore, Billerica, MA) and imaged with a confocal microscope (Zeiss LSM 700; Carl Zeiss Microscopy, Thornwood, NY).
Primary antibodies used for hFcRn detection were rabbit, polyclonal anti-FCGRT, Human Protein Atlas
14 (HPA)012122 (Sigma-Aldrich); HPA015130 (Sigma-Aldrich); M-255 (sc-66893; Santa Cruz Biotechnology, Dallas, TX); H-274 (sc-66892, Santa Cruz Biotechnology); mouse, monoclonal anti-FCGRT, 1G3 (myeloma hybridoma cell line CRL-2434); H-4 (sc-166413; Santa Cruz Biotechnology); and ADM31.
15 Iba-1 antibody (rabbit) was used as a macrophage marker.
16 Secondary antibodies (Invitrogen) used were goat AlexaFluor 594-conjugated anti-mouse IgG, donkey AlexaFluor 555-conjugated anti-rabbit IgG, rabbit anti-mouse IgG, and rabbit anti-AlexaFluor 488. Lectins used were fluorescein conjugated IB4 (Vector Labs, Burlingame, CA) for mouse and fluorescein conjugated UAE (Sigma-Aldrich) for human tissue. Conjugation of antibodies with AlexaFluor 488 was carried out using an antibody labeling kit (APEX; Invitrogen) according to the manufacturer's instructions.