Alexa 488 conjugated anti rabbit igg antibody

Cells were cultured on glass chamber slides at 37 °C overnight and exposed to deferoxamine (0, 100 μM) for 24 hours. Cells were then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with −20 °C methanol and 0.5% Triton X-100/PBS, and blocked with 10% fetal bovine serum and 2% bovine serum albumin in PBS. Cells were incubated with primary antibody for HIF-1α (#ab51608, Abcam, Cambridge, UK, 1:500) at 4 °C overnight. After washing by PBS twice, cells were incubated with Alexa 488-conjugated anti-rabbit IgG antibody (Invitrogen, CA, USA, 1:200) at room temperature for 10 minutes. After washing by PBS twice, the nuclei were stained with DAPI.

For measurement of the gut length in E18.5 embryos, the digestive tract was dissected out from 16 control and 6 Dlg1^-/- mice, and the length between the pylorus and the distal end of the rectum was measured. For analysis of embryos at an earlier stage, intestines were dissected out from 6 control and 11 Dlg1^-/- mice, and the enteric nerve plexus was stained by whole-mount immunohistochemistry with an anti-NGFR/p75 antibody (Promega, Fitchburg, WI, USA) and an Alexa 488-conjugated anti-rabbit IgG antibody (Invitrogen). Fluorescence images were captured by SteREOLumar.V12 and AxioCamMRm (Carl Zeiss), and the distances from the pylorus to the distal end of the NGFR/p75 signals, from the pylorus to the ileocecum (length of the small intestine), and from the ileocecum to the distal end of the rectum (length of the large intestine) were measured.

Mouse skin tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and sectioned. Sections were deparaffinized and stained with H&E for histopathological analysis. For immunohistofluorescence analysis, antigen retrieval was performed in sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) at 100 °C for 20 min, and blocked in 10% normal serum, 1% BSA, 0.1% Triton X-100 in PBS for 2 h at room temperature. Sections were incubated with anti-IL-17 antibody (Novus Biologicals, NBP1-72027, 1:100) and CCR6 antibody (Abnova, PAB12270, 1:250) overnight at 4 °C. After washing with PBS, sections were stained with Alexa 594 conjugated anti-Rat Ig G antibody (Invitrogen, A11007, 1:500) and Alexa 488 conjugated anti-Rabbit Ig G antibody (Invitrogen, A11008, 1:500) for 1 h at room temperature. Following PBS washing, sections were counterstained with DAPI, mounted in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, P36965), and analyzed under a confocal fluorescence microscope (Olympus, FV3000).
To measure keratinocyte proliferation in mouse skin tissue. 1 mg BrdU (Roche) was injected 12 h before killing. Fixed and paraffin embedded sections were deparaffinized and boiled in citrate buffer, and incubated with anti-BrdU antibody, followed by HRP-conjugated goat anti-mouse Ig G (Thermo Fisher Scientific, 31431, 1:5000)

Cells were cultured on 35-mm glass bottom dishes (Iwaki glass) for 24 h with or without 1 μg/ml doxycycline. For the confocal microscopy analysis of the expression of HRP, hmKR or hmAG, each expressed cells were treated with antibodies against HRP (Jackson ImmunoResearch), hmKR (MBL) or hmAG (MBL) at room temperature for 20 min. Then, the cells were treated with Alexa 488-conjugated anti-goat IgG antibody (Invitrogen) for HRP, Alexa 555-conjugated anti-mouse IgG antibody (Invitrogen) for hmKR and Alexa 488-conjugated anti-rabbit IgG antibody (Invitrogen) for hmAG at room temperature for 20 min. After washing with PBS, the cells were fixed with 7.4% formaldehyde-PBS solution at room temperature for 10 min. The cells were gently washed with PBS, and observed with confocal laser scan microscopy (FLUOVIEW FV1000, OLYMPUS).