Anti mouse cd3

Skin biopsies were collected from the dorsal side of SPF and GF mice, fixed in 10% (w/v) formalin, embedded in paraffin, and sectioned at 6 μm. Tissue sections were stained with hematoxylin and eosin to characterize epidermal thickness or with toluidine blue to identify mast cells. For immunofluorescence, sections were deparaffinized with xylene and rehydrated in downgraded alcohol. Heat-inactivated antigen retrieval was performed by incubating the tissue sections in 10-mM sodium citrate buffer, pH 6.0, and subsequently washing the sections with a PBS/0.2% Triton solution. Tissue sections were blocked with 10% (v/v) normal goat serum for 2 h at room temperature. After blocking, sections were incubated with a primary antibody. The antibodies that were used include anti-mouse Keratin 6A (Biolegend), anti-mouse Loricrin (Biolegend), anti-mouse CD3 (Abcam), and anti-mouse Ki67 (Abcam). Following multiple washes, secondary antibodies, goat anti-rabbit IgG-Alexa, and goat anti-mouse-Alexa 555 were applied for 1 h at room temperature and then washed. Slides were mounted with prolong DAPI (Molecular Probes) and examined under a fluorescent microscope (Leica DM550B). Positive-stained cells were counted in five fields per tissue section at × 400 magnification, three tissue sections per mouse, and three mice per group.

Kidney tissue samples were collected from all mice and fixed overnight in 4% buffered formalin. Subsequently the fixed tissue samples were embedded in paraffin and cut into 4 µm thick serial sections. Kidney paraffin sections were stained with periodic acid-Schiff (PAS) reaction using standardized protocols as described^{41 (link)}. Scoring of lupus nephritis activity and chronicity indices was performed according to human lupus nephritis^{42 (link), 43 (link)}. Methenamine silver staining was performed on paraffin embedded kidney sections and semiquantitatively scored form 0–3 by a blinded observer. For immunostaining the following primary antibodies were used: anti-mouse CD3 (Abcam, Cambridge, UK), complement C3c (Biobyt Ltd, Cambridge, UK), CD31 (Dianova GmbH, Hamburg, Germany), WT1 (Santa Cruz Biotechnology, Dallas, Texas) and nephrin (Acris Antibodies, San Diego, CA). The immunostaining for C3c and CD31 were analyzed using the ImageJ software by measuring positively stained area and dividing it by the total area of the glomerulus and reported as percentage of +ve area. Sections stained for WT1/nephrin and CD3 were evaluated by counting positive cells per glomerulus. All the morphometric analysis were carried out in a blinded fashion.

Non-perfused lungs from unvaccinated and MIP-vaccinated mice were fixed in 4% (wt/vol) paraformaldehyde for 24 h and then dehydrated and embedded in paraffin for analysis. Sections (4 μm) were taken on glass slides, deparaffinized, and subjected to immunofluorescence. Tissue sections were stained with antibodies against T-cells (anti-mouse CD3; Abcam, Cambridge, UK) and B-cells (biotin-conjugated B220; BioLegend, San Diego, CA, USA). The secondary antibodies used were anti-rabbit Alexa Fluor™ 488 and streptavidin-conjugated Alexa Fluor 594 (Life Technologies, Carlsbad, CA, USA). Slides mere stained with DAPI (Sigma–Aldrich, Saint Louis, USA) and mounted using ProLong® Gold mounting reagent (Invitrogen, Carlsbad, CA, USA).