Hrp conjugated anti rat igg

The antigen of antibody 0614 was confirmed by Western blotting. Recombinant CD73 with a C-terminal His-tag was loaded onto SDS-PAGE gel, separated, and then transferred to PVDF membranes (Bio-Rad). Membranes were blocked for 5 min with Bullet Blocking One for Western blotting (Nacalai Tesque). The membranes were washed three times with TBS-T. The blots were then incubated with antibody 0614 (10 µg/mL) or anti-His tag antibody (1:10,000, ProteinTech, Rosemont, IL, USA) at room temperature for 1 h. The membranes were washed three times with TBS-T and then incubated with HRP-conjugated anti-rat IgG (1:5000; Jackson Immuno Research, West Grove, PA, USA) or anti-mouse IgG (1:5000; CST, Danvers, MA, USA) at room temperature for 1 h. Can Get Signal solution (Toyobo, Osaka, Japan) was used to reduce background noise. After the membranes were washed three times with TBS-T, proteins were visualized using ECL prime (GE Healthcare).

For whole cell samples, DT40 cells were harvested, washed with PBS, and suspended in 1xLSB (Laemmli sample buffer) (final 1 × 10⁴ cells/μl), followed by sonication and heating for 5 min at 96 °C. Proteins were separated on SuperSep Ace, 5–20% (Wako) and transferred to Immobilon-P (Merck) using HorizeBLOT (ATTO). Primary antibodies used in this study were rabbit anti-chicken CENP-T (Hori et al. 2008 (link)), rabbit anti-chicken CENP-H (Fukagawa et al. 2001 (link)), rabbit anti-chicken Dsn1 (Hara et al. 2018 (link)), rabbit anti-GFP (MBL), rat anti-RFP (Chromotek), and mouse anti-α-tubulin (Sigma). Secondary antibodies were HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch), HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch), and HRP-conjugated anti-rat IgG (Jackson ImmunoResearch). To increase sensitivity and specificity, Signal Enhancer Hikari (Nacalai Tesque) was used for all antibodies. The antibodies were incubated with the blotted membranes for 1 h at room temperature or for overnight at 4 °C. Proteins reacting with antibodies were detected with ECL Prime (GE Healthcare) and visualized with ChemiDoc Touch (Bio-Rad). Acquired images were processed using Image Lab 5.2.1 (Bio-Rad) and Photoshop CC (Adobe).

Rat spinal cord was processed as previously described (31 (link)) using as primary antibodies the IgGs contained in sera samples or commercial antibodies against MBP (1:1,000, Covance, Princeton, NJ, USA), NFM (1:500, Aves Lab, Tigard, Oregon, USA), GFAP (1:2,000, Dako, Glostrup, Denmark) and APC (1:300, Calbiochem, Darmstedt, Germany). To detect these antibodies we used Cy3 conjugated anti-human IgG (1:1,000, Jackson Immunoresearch, Ely, UK), Cy5 anti-chicken IgY, Alexa Fluor-594 anti-rabbit IgG (1:1,000, Invitrogen, Barcelona, Spain) and Alexa Fluor-488 anti-mouse IgG (1:1,000, Invitrogen). To detect endogenous IgG in rat spinal cord, HRP-conjugated anti-rat IgG (1:1,000; Jackson Immunoresearch) was used and their localization was detected by DAB-peroxidase reaction. Also, HRP-conjugated anti-rabbit IgG (1:1,000; Jackson Immunoresearch) was employed as a control of specificity. When performed, nuclei were counterstained with Hoescht 33,258 (1:5,000; Invitrogen).
Samples were analyzed with a LEICA SP5 confocal microscope. All post-capture image modifications were identically performed between groups, including cropping, noise reduction and minor adjustments to optimize contrast and brightness.