Ab97234

For IHC staining, formalin-fixed and paraffin-embedded small intestine sections were incubated with primary antibodies against IgA (Abcam, ab97234) and analyzed using streptavidin peroxidase detection system (Maixin) according to the manufacturer’s protocol. DAB (Maixin) was used as an HRP-specific substrate. Staining intensity for IgA was ranked using a 5-point scale from 0 (unstained) to 4 (very intensively stained). The histopathological evaluations were performed by three independent pathologists. Every group and tissue examination were blinded.
The kidney and small intestine tissue for IF staining was cut into frozen sections and fixed with acetone for 1 min. After fixation, they were blocked with 2% bovine serum albumin diluted by PBS at room temperature for 1 h. They were washed with PBS three times and incubated with FITC-labelled goat anti-mouse IgA (Abcam, ab97234) at 37°C for 40min. After washing with PBS three times, they were mounted with anti-quenching tablets and observed under a confocal microscope. Cell nuclei were stained with DAPI. Images were quantified by counting the number of positive nuclei and divided by the total number of nuclei.

Combined in situ hybridization with immunofluorescence was carried out by first doing the in situ protocol as described above. After the last wash in PBS, the slides were then re-fixed in 4% PFA for 1 h at room temperature. The immunofluorescence protocol was then carried out as described above except 1 mg/ml UltraPure BSA (Thermo Fisher Scientific, AM2616) was used in the blocking and antibody incubation steps. The primary antibody used was KT3 (DSHB; 1:100). The secondary antibody used was goat anti-mouse IgA conjugated to FITC (Abcam; ab97234; 1:500).

Sections were cut at 12 μm. For mice, control and mutant sections were arranged side-by-side on the same slide to share the same antibody pools. Sections were permeablised and blocked using 3% BSA in PBS+0.3% Triton X100 for 1 h. Sections were incubated for 2 h at RT or overnight at 4°C with primary antibodies against BAG3 (as described previously; 1:200), FLNC (RR90; mouse IgA monoclonal, kind gift from Peter van der Ven, University of Bonn; 1:20), S58 (mouse IgA monoclonal against slow myosin MYH7B, Developmental Studies Hybridoma Bank, DSHB; 1:10), F59 (mouse IgG monoclonal against all fast isoforms, DSHB; 1:10) or ubiquitin (P4D1, a mouse IgG monoclonal, Cell Signaling; 1:200) in blocking buffer followed by three washes with PBS for 5 min. Slides were then incubated for 1 h with appropriate combinations of compatible secondary antibodies at RT in the dark (e.g. anti-Rabbit IgG TRITC; ab6718; 1:70 and anti-Mouse IgA FITC; ab97234; Abcam; 1:70) before three washes with PBS for 5 min. Slides were mounted with Mowiol (Sigma) plus DAPI to stain nuclei. Images were obtained with exposure times of 20 ms (DAPI), 200 ms (FITC) and 300 ms (TRITC), using a Leica DMIL LED microscope plus a Leica DFC 3000 G camera and the Leica LAS X software.