Hrp conjugated donkey anti rabbit igg

Tissues were fixed in 4% (w/v) paraformaldehyde at 4 °C overnight and embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan). To analyze the localization of macrophages, immunohistochemistry was performed on frozen tissue sections (10 μm). Specifically, sections were treated with REAL Target Retrieval Solution (DAKO, Carpinteria, CA, USA) at 98 °C for 40 min to retrieve the antigen and incubated in 3% H₂O₂ at room temperature (RT) for 10 min to inhibit endogenous peroxidase. After washing, sections were incubated with 0.5% (w/v) blocking reagent (PerkinElmer, Waltham, MA, USA) at RT for 30 min to block the non-specific binding of antibodies and then treated with primary antibodies at 4 °C overnight followed by secondary antibodies at RT for 1 h. The primary antibodies used in the present study were rabbit anti-CD11c (clone D1V9Y, 1:100, Cell Signaling Technology, Danvers, MA, USA) and rat anti-F4/80 (clone BM8, 1:50, BioLegend). Secondary antibodies were horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor 647-conjugated donkey anti-rat IgG (1:200, Jackson ImmunoResearch). The enzyme activity of HRP was visualized using the TSA Plus fluorescein system (PerkinElmer). Sections were then counterstained with DAPI. Tissue images were obtained using a BZ-9000 microscope (Keyence, Osaka Japan).

Total cell extract was quantitated for the amount of protein using a BCA Protein Assay Kit (Thermo Fisher Scientific). About 10 μg of protein was loaded in each well of 15 per well NuPAGE Bis‐Tris gels, run with XCell SureLock Mini‐Cell Electrophoresis System and transfer to nitrocellulose membrane with XCell II Blot Module (Thermo Fisher Scientific). For immunolabelling, primary and secondary antibodies were incubated overnight diluted in TBS + 5% milk. Antibody labelling was revealed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and visualised in a G:BOX Chemi XT4 machine (Syngene, Cambridge, UK). Antibodies used: rabbit polyclonal anti‐IMPDH2 (1 : 1000, ProteinTech, Chicago, IL, USA; 12948‐1‐AP); HRP‐conjugated mouse monoclonal anti‐ACTB (1 : 3000, ProteinTech, HRP‐60008). HRP‐conjugated donkey anti‐rabbit IgG (1 : 1000, Jackson ImmunoResearch, Cambridgeshire, UK; 711‐035‐152).

Cells were resuspended in Thorner buffer with an equal volume of glass beads (Sigma Cat. # G8772) and vortexed for 10 min at room temperature. Equal amounts of protein were loaded into each lane and run on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (BioRad; Cat. #162–0115). Membranes were blocked with 5% milk, incubated with rabbit anti-HA (AbCam; Cat. # ab9110) at 1:4,000 or mouse anti-Vma2 (Life Technologies; Cat.# A-6427) at 1:4000, followed by HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Labs; Cat. # 711-035-152) at 1:10,000 or HRP-conjugated donkey anti-mouse (Jackson ImmunoResearch Labs; Cat. # 715-035-151) at 1:10,000. Membranes were developed using Clarity Western ECL Substrate (BioRad; Cat. # 170–5060) and imaged using a FujiFilm LAS-3000.

MDCK, CHO^GFP-rMAL, CHO^GFP and mock transfected CHO cells (CHO^mock) cells were grown in 6- well plates (Costar) until confluence. Activated ETX (60nM) or vehicle control was administered to cells and allowed to incubate for 1 hour at 37 C°. Cells were detached from plate following trypsin digestion. Cells were collected, washed 3 times in PBS and re-suspended in PBS containing 0.2% SDS. Harvested cells were then triturated with a serological pipette followed by trituration with an insulin syringe until DNA was sufficiently sheared. ETX complex containing detergent resistant membranes was isolated by centrifugation at 14, 000G for 5 minutes and collecting the pellet. The pellet was washed 3 times in PBS and re-suspended in 100 ul PBS. An equal volume of 2 X Lamelli sample buffer (Bio-rad) was added to each sample for SDS page electrophoresis. Proteints were transferred to an Immobilon-P membrane (Millipore) and probed with a custom rabbit anti-ETX antibody generated against the N terminal of ETX sequence KASYDNVDTLIEKGR (Pacific Immunology). HRP conjugated donkey anti-Rabbit IgG (Jackson Immunoresearch) was used to visualize the ETX complex.