Immunoprecipitation was performed with protein G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) according to the standard procedures. Briefly, cells after transfection were incubated in IP lysis buffer for 30 min at room temperature. The extracts were incubated with anti-LeY-antibody (2 μg/mL), and the protein G agarose beads were incubated with goat anti-mouse IgM (62-6820, Invitrogen) (1:100) at 4 °C overnight. The beads were washed for three times with washing buffer and the immunoprecipitants were purified by protein G agarose beads with gentle rocking for 3 h. Finally, the beads were washing for five times with washing buffer and added in 50 μl SDS-loading buffer. The whole cell lysates were incubated at 70 °C for 15 min followed with western blot analysis.
Goat anti mouse igm
Manufactured by Thermo Fisher Scientific
Goat anti-mouse IgM is a secondary antibody used in various immunoassay techniques. It is specific for the mouse IgM isotype and can be used to detect or quantify mouse IgM in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Ab7817, by Abcam (2 mentions)
Goat antipecam 1 antibody sc 1506, by Santa Cruz Biotechnology (2 mentions)
D1306, by Thermo Fisher Scientific (2 mentions)
Protein g agarose bead, by Thermo Fisher Scientific (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
Tunel assay kit, by Roche (1 mentions)
Goat anti mouse igg, by Southern Biotech (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Albumin fraction 5, by Carl Roth (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boehringer Ingelheim (1 mentions)
Asp300, by Leica (1 mentions)
Donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
As1842856, by MedChemExpress (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
Anti cd40, by Thermo Fisher Scientific (1 mentions)
10 protocols using goat anti mouse igm
1
Immunoprecipitation of LeY-antibody Complexes
2
Assessing Cell Proliferation and Apoptosis
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Cell proliferation was assessed by immunofluorescence staining of paraffin-embedded sections with antibodies to pH3 (1:100) and Ki67 (Abcam, #ab15580, 1:500). Secondary antibodies included goat anti-rabbit IgG (Invitrogen, #A21429, 1:1,000) and goat anti-mouse IgM (Invitrogen, #A21042, 1:1,000). DAPI staining was used for visualization of cell nuclei and cells in anaphase or telophase versus prometaphase or metaphase. Apoptosis was analyzed with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) with a TUNEL assay kit (Roche, #12156792910). Sections were imaged with confocal microscopy and quantitatively analyzed with ImageJ.60 (link) All primer sequences are provided in Table S5 .
3
Immunoglobulin and Anti-dsDNA Antibody Quantification
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Serum samples obtained from all mice groups at different age intervals were tested for the presence of total immunoglobulins and anti-dsDNA antibodies using ELISA assays. In the case of total immunoglobulin detection, microplates were coated with 2 µg/ml goat anti-mouse IgG (Southern Biotech) or IgM. Following blocking, serially diluted immunoglobulin (Ig) standards (IgG, Catalog# I5381, Sigma-Aldrich; mouse IgM, catalog# 010-0107, Rockland) or serum samples were added into the microplate. After a two-hour incubation at room temperature, the bound Igs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Catalog# 2040-04, Southern Biotech) or goat anti-mouse IgM (Catalog# 626822, Invitrogen). Subsequently, quantification was carried out by measuring the absorbance at 405 nm. For the detection of IgG anti-dsDNA antibodies, 100 μg/ml of mBSA in phosphate-buffered saline (PBS) was applied to pre-coat the plates at 37°C for thirty minutes prior to the incubation of dsDNA standards (Catalog# D1501, Sigma) with an initial concentration at 1250 ng/ml. Serum samples were diluted 1:50 in PBS and added to the plates for a two-hour incubation. Alkaline phosphatase-conjugated goat anti-mouse IgG was used as the detection antibody.
4
Immunohistochemical Analysis of Protocadherins
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Wild-type C57BL/6J and homozygous Pcdha9 mutant embryos were obtained at E14.5. The embryos were fixed in 4% paraformaldehyde and processed for cryoembedding. Frozen sections were collected for immunostaining. Cryosections of mouse embryonic heart tissue were immunostained with rabbit anti-alpha-protocadherin antibody (190003, Synaptic Systems, 1:500), goat antiPECAM-1 antibody (sc-1506, Santa Cruz Biotechnology, 1:500), and mouse anti-alpha smooth muscle actin antibody (1A4) (ab7817, Abcam, 1:500). Human adult aortic tissues were stained with the alpha-protocadherin antibody and also imaged for elastin autofluorescence. Secondary antibodies included goat anti-rabbit IgG (Invitrogen, #A21429, 1:1,000) and goat anti-mouse IgM (Invitrogen, #A21042, 1:1,000). DAPI was used to label nuclei (D1306, Invitrogen). Sections were imaged with confocal microscopy and quantitatively analyzed with ImageJ.40 (link)
5
Formalin-Fixed Paraffin-Embedded Tissue Immunohistochemistry
6
Immunostaining of Pcdha9 Mutant Embryos
7
Immunofluorescence Staining of Leishmania Parasites
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Parasites were fixed with 2% formaldehyde in 10 mM phosphate buffer, pH 7.2, containing 150 mM NaCl (PBS), for 10 min. Some fixed cells were permeabilized with 0.1% saponin. Cells were washed, resuspended in 1 ml PBS, and 100 μl of the solution was added to coverslips pretreated with 0.1% poly-L-lysine. Coverslips were blocked for 3 h with 0.1% gelatin in PBS and for 1 h with 10% skimmed milk and 1% bovine serum albumin (BSA) in PBS. Parasites were incubated sequentially with primary antibody LST-1 (mouse IgM; reacts with Leishmania IPC; Godoy et al., MS in prep) and SST-1 (IgG3; reacts with L. braziliensis promastigote glycolipids) (Silveira et al., 2005 (link)) for 1 h, washed with PBS, and incubated with goat anti-mouse IgM (μ chain) conjugated to Alexa Fluor 647 or with goat anti-mouse IgG (gamma chain) conjugated to Alexa Fluor 488 (Thermo Fisher) in a solution containing 1% BSA and 0.01 mM 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) in PBS. Slides were examined under conventional or confocal fluorescence microscopy with a Leica SP5 TS system with a 100x/1.44 oil objective. Images were acquired under optimal instrument settings and processed by the ImageJ program (http://rsbweb.nih.gov/ij/ ).
8
Assessing IL-21 Regulation of B Cell Signaling
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Splenic CD19+ B cells from BALB/c mice were purified using magnetic mouse CD19 MicroBeads (Miltenyi Biotec) and cultured in flat-bottom 96-well plates (Corning). To assess effects of IL-21 on the expression of c-Myc, BATF, IRF4, p-S6, cyclin D3, and Foxo1, cells were incubated with the indicated concentration of IL-21 (PeproTech) and 20 μg/ml of anti-mouse CD40 (FGK4.5) for 24 h at 37°C. To inhibit Foxo1, cells were treated with AS1842856 (2 μM; MedChem Express) for 1 h, followed by further incubation with 20 μg/ml of anti-CD40 mAb with or without 80 ng/ml of IL-21 for 16 h. To study Foxo1 intracellular localization, cells were rested for 30 min at 37°C, and where indicated, further incubated with 10 μg/ml of goat anti-mouse IgM (μ chain specific; Thermo Fisher Scientific) for 10 min followed by a 30-min stimulation with 80 ng/ml of IL-21.
9
Immunostaining and Imaging of Oligodendrocytes
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The cultured cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) and used for immunostaining. Fixed cells were blocked with 5% normal goat serum in phosphate-buffered saline and 0.1% Triton X-100 (PBST) and then incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: rat anti-GFP antibody (1/500, Nacalai Tesque, 04404-84), monoclonal anti-α-tubulin antibody (1/500, Sigma, T9026), rat anti-myelin basic protein (MBP) antibody (Millipore, MAB386), monoclonal O4 antibody (1/300, R&D systems, MAB1326) and rabbit anti-paxillin antibody (1/250, abcam, ab32084, clone Y113). After being rinsed with PBST, the cells were incubated with secondary antibodies. The secondary antibodies used were Alexa 488- or 594-conjugated goat anti-mouse, anti-rabbit and anti-rat IgG or goat anti-mouse IgM (Molecular Probes). Fluorescent signals were visualized using AX70 fluorescence microscope (Olympus, Tokyo) and A1Rsi confocal fluorescence microscope (Nikon, Tokyo). The number of OL primary processes was analyzed using “Analyze/Sholl” tool of Fiji software based on ImageJ (NIH).
10
Whole Mount Embryo Immunostaining Protocol
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Embryos were harvested in 1X PBS and fixed in 4%PFA in PBS at 4°C overnight. Standard whole embryo immunostaining was performed as previously described (Inman et al., 2013 (link); Sandell et al., 2014 (link)). Primary antibodies against the following proteins were used: β-tubulin III (TuJ1) at 1:1000 dilution (Covance Research products, cat# MMS-435P), PECAM1/CD31 at 1:400 dilution (BD Pharminogen, cat# 553370), cleaved caspase 3 at 1:1000 dilution (Cell Signaling Technology, cat# 9661S) and Sox10 at 1:500 dilution (Abcam, cat# 155279). Secondary antibodies used were: Goat anti-Mouse IgM (Invitrogen/Molecular Probes, cat# A-21042), Goat anti-Rabbit IgM (Invitrogen/Molecular Probes, cat# A-11034) and Peroxidase AffiniPure Donkey Anti-Rat IgG (H + L) (Jackson ImmunoResearch, cat# 712-035-153) at 1:500 dilution. Immunostained embryos were mounted in Vectashield Antifade Mounting Media (Vectorlabs, cat# H-1000) and imaged using an upright confocal microscope. Except for Pecam1 staining in Figure 1 , control and mutant embryos were imaged with the same magnification.
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