Peroxidase conjugated goat anti mouse igg

Tissues were fixed in 10% neutral-buffered formalin for 7 days and then decalcified in 14% ethylenediaminetetraacetic acid for up to 4 months. After decalcification, samples were embedded in paraffin, coronally sectioned (5 μm), standard Alcian Blue Hematoxylin was performed to assess repair tissue. For immunohistochemistry, samples were pretreated (hyaluronidase 200 U mL⁻¹) for one hour at 37 °C. Sections were blocked with 10% Normal Goat Serum in Tris Buffered Saline (TBS) and subsequently treated with a primary antibody against collagen type II (1:100, Iowa hybridoma bank) overnight at 4 °C. The secondary antibody, peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch), was diluted 1:500 and incubated at room temperature for 1 h. Slides were developed using the DAB substrate kit (Vector Laboratories). Negative controls were prepared by omitting the primary antibody. Slides were digitized with the Axio Scan.Z1 (Zeiss).

Anti-pneumococcal IgG titers were assessed in serum from uninfected and infected mice using whole bacterial cells as the capture antigen, as described previously with some adjustments [28 (link)]. Type 23F strain was grown to an OD₆₂₀ of 1 and washed with PBS. Pneumococcal cells were diluted to final OD₆₂₀ of 0.1 in coating buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃). Microtiter plates (2HB, Immunolon) were coated with 100 μL suspension/well at 4°C overnight. The next day, the plates were blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 1 hour, after which the plates were incubated with serial serum dilutions in PBS at 4°C overnight. Peroxidase-conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories) was applied and plates were incubated at 37°C for 1.5h. Between incubation steps, plates were washed three times with 0.05% Brij-35 (Thermo Fisher Scientific) in PBS. Plates were developed using 100 μL/well substrate o-phenylenediamine dihydroxychloride (OPD, Thermo Fisher Scientific, 1 tablet in 7.5 ml H₂O with 7.5 μL 30% H₂O₂) and incubated at room temperature for 30 minutes in the dark. Reactions were stopped with 50 μl/well of 2M H₂SO₄ and the absorbance was measured at 492 nm. Serum IgG titers were determined by calculating the dilution at which the absorbance was equal to an OD₄₉₂ of 0.1.

Cell lysates and proteins eluted from co-immunoprecipitations were resolved by 12% SDS-PAGE, transferred to PVDF membrane (Bio-Rad), and probed with antigen-specific primary antibodies. The following primary antibodies were used for Western Blot detection: rabbit polyclonal anti-GFP (Molecular Probes), rat monoclonal anti-RFP (clone 5F8, Chromotek), mouse monoclonal anti-Rab10 (Sigma), and mouse monoclonal (6C5) anti-GAPDH (Millipore).
Blocking was performed with 5% skim milk. For all analyses, HRP-conjugated secondary antibodies were used (peroxidase-conjugated goat anti-rabbit IgG (Sigma), peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch) or peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch)) and detection was performed using ECL detection system (Luminata Classico Western HRP substrate (Millipore) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo)).

The following primary antibodies were used: Mouse monoclonal anti-FLAG [1:1,000 (IF), 1:20,000 (WB), Sigma], mouse monoclonal anti-Myc [1:1,000 (IF), 1:1,000 (WB), Cell Signaling], rabbit polyclonal anti-Actin (1:1,000, Sigma), rabbit polyclonal anti-tRFP (1:2,000, Evrogen), and rabbit polyclonal anti-IncA (1:200, kind gift from T. Hackstadt, Rocky Mountain Laboratories). The following secondary antibodies were used: Peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Jackson ImmunoResearch), peroxidase-conjugated goat anti-mouse IgG (1:10,000, Jackson ImmunoResearch), goat anti-mouse AlexaFluor 488 or 514 (IF: 1:1,000, Molecular Probes), and goat anti-rabbit Pacific Blue (IF: 1:1,000, Molecular Probes).

For all immunoblotting experiments, cells were grown in six-well plates and used at 75% confluency for treatments. After the indicated treatments, the cells were washed with ice-cold PBS and lysed using RIPA lysis buffer, supplemented with protease–phosphatase inhibitor (MS-SAFE; Sigma-Aldrich). Protein concentration was determined using Bio-Rad DC protein assay (Bio-Rad laboratories). 30 μg cellular protein lysates were resolved by 12% SDS–PAGE, transferred to PVDF membrane (Bio-Rad), and probed with antigen-specific primary antibodies. Blocking was performed with 5% skim milk; except for the phospho antibodies, which was instead performed with 5% bovine serum albumin. For all analyses, HRP-conjugated secondary antibodies were used (peroxidase-conjugated goat anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch)) and detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The results were analyzed using Image Lab v6.1 by Bio-Rad laboratories.

The following primary antibodies were used for immunofluorescence (IF) microscopy and Western blotting (WB): mouse anti-CT147 (kind gift from Guangming Zhong, UTHSCSA) (1:50 for IF), mouse monoclonal anti-FLAG (1:1,000 for IF; 1:10,000 for WB; Sigma), rabbit polyclonal anti-GFP (1:2,000 for WB; Invitrogen), rabbit polyclonal anti-mCherry (1:2,000 for WB; BioVision), rabbit polyclonal anti-HA (1:300 for IF; Sigma). The following secondary antibodies were used: Alexa Fluor 594-conjugated goat anti-mouse antibody, Alexa Fluor 514-conjugated goat anti-mouse antibody, Alexa Fluor PB-conjugated goat anti-rabbit antibody (all 1:500 for IF; Molecular Probes), peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (both 1:10,000 for Western blotting; Jackson ImmunoResearch).