Goat anti mouse igg antibody

After euthanasia, hearts were removed and snap-frozen at -80°C. Axial cryosections were cut to a thickness of 7 μm for subsequent immunohistochemistry. Every second cut was stained with haematoxylin-eosin (H.E.). Inflammatory cells were detected by specific antibodies using the labelled streptavidin-biotin method. Briefly, anti-CD68/ED1 (dilution 1 : 50; Santa Cruz Biotechnology Inc., Santa Cruz, USA) was used to detect macrophages as primary antibody. A goat anti-mouse IgG antibody (dilution 1 : 500; Jackson Immunoresearch Laboratories Inc., West Grove, USA) was used as a secondary antibody. Images were acquired with an Olympus BX61 microscope equipped with a CCD-camera (ColorView II, Olympus Germany). Quantification of CD68/ED1 positive cells was performed using Cell-F software (Olympus, Hamburg, Germany), measuring positively stained areas in μm² using color threshold values [19 (link), 20 (link)].

Paraffin sections (5 µm) of normal tissue samples were obtained from CMMI-DIAPath (Belgium) and Biobanque Hôpital Erasme-ULB (BE_BERA1, Belgium). Sections were deparaffinized, rehydrated, microwaved for 20 min in citrate buffer (10 mM sodium citrate pH 6.0, 0.05% Tween 20) for antigen retrieval, and treated for 20 min with 3% hydrogen peroxide to block endogenous peroxidase. The sections were incubated for 30 min with 5% heat-inactivated normal serum and further with 300 nM of CA7281 nanobody diluted in PBS/1% heat-inactivated normal serum for 1 h at 4°C. After a short washing step, bound nanobodies were fixed by a 20 min treatment with 2% paraformaldehyde solution. Bound nanobodies were detected with successive 30 min incubations with a mouse anti-His Tag mAb (Bio-Rad; 1/2,000), a goat anti-mouse IgG antibody (Jackson; 1/100) and mouse peroxidase-antiperoxidase complex (Jackson; 1/400). After washing with PBS, sections were incubated with ImmPACT DAB Substrate (Vector Laboratories) and monitored under a microscope. Reaction was stopped by immersion of the slides in distilled water. The slides were counterstained with hematoxylin and eosine, dehydrated, coverslipped, and examined by light microscopy (Axioplan 2, Zeiss).

The anti-ζ-globin chain mAbs PL2 (IgG1 isotype) and PL3 (IgG1) [28 (link)] and the mouse anti-Ag85B mAb clone AM85B-8B (IgG1) [29 (link)] were generated in our laboratory. Goat anti-mouse IgG antibody was obtained from Jackson ImmunoResearch (West Grove, PA, USA). EZ-Link^™ Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, IL, USA). Horseradish peroxidase (HRP)-labeled streptavidin and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate were purchased from Invitrogen (Camarillo, CA, USA). Goat anti-mouse immunoglobulins antibody was obtained from KPL (Gaithersburg, MD, USA). The IC strip test for the determination of Hb Bart’s in RBC hemolysates was purchased from i+Med Laboratories Co., Ltd. (Bangkok, Thailand).

U-2 OS cells with stable expression of FUCCI Cell Cycle Sensor^{36 (link)} were analyzed on BD Influx cell sorter to obtain samples of 2 × 10⁵ cells double-negative for Cdt1-RFP and geminin-GFP (early G1 phase) and corresponding samples of Cdt1-RFP-positive/geminin-GFP-negative cells (more advanced G1 phase). One set of such samples (referred to as “total samples”) was immediately extracted in sample buffer (15 mM Tris, pH = 6.8, 0.5% SDS, 2.5% glycerol, 1.25% 2-Mercaptoethanol, traces of bromophenol blue) and processed for SDS PAGE/Western blot to allow standardization to total levels of tubulin (using anti-α-Tubulin antibody, cat. no. T9026, Sigma, and Goat Anti-Mouse IgG antibody, 115-035-146, Jackson Immunoresearch). The second set was resuspended in 200 µL cold nuclei extraction buffer (320 mM sucrose, 5 mM MgCl₂, 10 mM HEPES, 0.25% Triton X-100, pH 7.4), incubated for 10 min on ice, centrifuged at 2000 × g and the pellet was washed twice with phosphate-buffered saline (PBS) to yield clean nuclear samples. Protein samples from nuclei were then extracted and processed as described for total samples. Purity of the nuclear isolation was independently verified using antibodies against cytosolic protein AKT1 (Cell Signaling, #2938) and mitochondrial inner membrane protein SDHA (Abcam, ab14715) as negative controls (data not shown).

Extracted protein samples (20 μg per well) were separated on a 12% SDS-Bis-Tris polyacrylamide gel. After transfer, the nitrocellulose membrane was incubated over night with 10 mL of primary antibodies against PARP-1, LMNA (Santa Cruz Biotechnology, Delaware Avenue, CA, USA), and β-actin (Santa Cruz Biotechnology) at 4°C. The membrane was then incubated with the appropriate goat anti-mouse IgG antibody (1 : 1000) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) to detect biotinylated protein markers in 10 mL of blocking buffer with gentle agitation for 1 hour at room temperature. The proteins were visualized using a chemiluminescence detection system (Amersham ECL system, London, UK).

Cell lines or PBMCs (1 × 10⁵/100 µL) were pre-incubated with mAb MT99/3 at a final concentration of 20 µg/mL or isotype-matched control mAb 4G2 or medium (No Ab) for 30 min at room temperature. The treated cells were centrifuged and the excess mAbs were removed. The secondary antibody crosslinker goat anti-mouse IgG antibody (#115-005-072 Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 5 µg/mL, 150 µL were added into the treated cells and incubated for 30 min at room temperature. After that, the activated cells were incubated at 37 °C in a humidified 5% CO₂ incubator for 0 min, 90 min and 180 min. Treated cells were harvested and stained with apoptosis markers Annexin V-FITC (BioLegend, San Diego, CA, USA) and 7-AAD solution (BioLegend). Cell apoptosis was determined by a BD Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software V10.6.2.