2.7. Immunolocalization and Western Blot Analysis
This protocol is extracted from research article:
RedEfish: Generation of the Polycistronic mScarlet: GSG-T2A: Ttpa Zebrafish Line
Antioxidants (Basel), Jun 16, 2021; DOI: 10.3390/antiox10060965

Distribution of mScarlet protein was assessed in embryos (3 dpf) using the polyclonal antibody to red fluorescent protein (anti-RFP) (anti-rabbit RFP, 1:3000; abcam, ab62341 (Cambridge, MA, USA)). Monoclonal antibodies generated against acetylated tubulin (mouse anti-goat, 1:4000; Sigma Aldrich (St. Louis, MO, USA)) were used to label axons in the developing embryo as a positive control. Embryos (n = 15 per group) at the described time points were fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), washed in PBS the following morning and stored at 4 °C in PBS and 0.1% sodium azide until use. Fixed embryos were washed in PBS + 0.1% Tween20 (PBST), permeabilized with 0.005% trypsin in PBS on ice for 5 min, rinsed with PBST and post-fixed in 4% PFA. Permeabilized embryos were blocked in 10% normal goat serum in PBS + 0.5% Triton X-100 for an hour followed by incubation with the primary antibody overnight at 4 °C in 1% normal goat serum in PBS and 0.5% Triton X-100. Embryos were washed 4 times for 30 min in PBST, incubated with secondary antibody (Alexa-488 goat anti-mouse, 1:1000; ThermoFisher (Carlsbad, CA, USA)) for 2 h and washed 4 times for 30 min in PBST.

Distribution of Ttpa protein was assessed in zebrafish embryos (5 dpf) using the polyclonal anti-TTPA antibody (rabbit anti-zebrafish Ttpa (L6308, Antibodies Inc., Davis, CA, USA), 1:300). Embryos previously fixed in 4% PFA were washed in PBST and stored in 100% methanol. Samples were rehydrated and treated with 10 mg/mL proteinase K in PBST. Samples were then blocked in 5% normal goat serum and 3 mg/mL bovine serum albumin in PBST followed by incubation with the primary antibody. Embryos were washed, incubated with secondary antibody (Alexa-594 goat anti-rabbit, 1:1000; ThermoFisher (Carlsbad, CA, USA)), washed and visualized, as described [42].

All fixed embryo imaging was conducted using Keyence BZ-x700 All-in-one microscope with a 2×, 10× or 20× objective and 470 nm or 694 nm filter, as described above. Image adjustments, including cropping, brightness and contrast were performed uniformly using Adobe Photoshop.

For Western blots, protein was extracted from pooled (n = 25) embryos (24 hpf) in RIPA buffer supplemented with protease and protein phosphatase inhibitors (Calbiochem, La Jolla, CA, USA). Rat liver extract and extracts of c269 embryos were used as positive controls for Ttpa and RFP. Extracted protein (20 μg) was subjected to SDS-Page and processed by immunoblotting. Antibodies used for Western blotting were anti-ATP synthase F1 subunit α (1:1000, ab110273), anti-Ttpa (1:250, L6308), anti-RFP (1:1000, ab62341), goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP secondary antibodies (1:10,000). Nonspecific protein binding was blocked using Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% nonfat dry milk. All antibodies were diluted in TBST containing 1% bovine serum albumin. Proteins were visualized by SuperSignalTM West Femto Chemiluminescent Substrate (ThermoFisher, Carlsbad, CA, USA) and quantified using the Bio-Rad Image System (Hercules, CA, USA).

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