Zebrafish Husbandry. This study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) protocols AC18-014 and AC18-15 at Boise State University. Zebrafish (AB strain purchased from Zebrafish International Resource Center (ZIRC), University of Oregon, Eugene, Oregon, USA; zebrafish.org) were housed under standard laboratory conditions [26]. Developmental staging was reported as hours post-fertilization (hpf) at 28.5°C. Embryos were raised to 24–72 hpf in egg water (pH 7.2) [26]. Heterozygous CRISPR/Cas9-generated knockout of Col11a1a was lethal in the majority of offspring [18]; therefore, heterozygous crosses were used to generate embryos in addition to wildtype to validate the antibody. All embryos were humanely euthanized at 24–72 hpf on ice for 30 minutes followed by treatment with tricaine for 10 minutes, frozen and then fixed in 4% paraformaldehyde (PFA) as described below. No animals were excluded during this study. Potential confounders were not controlled. A total number of 105 embryos were used in this study, the minimum number to allow detection of protein.
Antibody design and development. Antibodies were generated using the peptide sequence NH2-ck(g)9dvphkdtlqa-COOH conjugated to keyhole limpet hemocyanin. Custom primary antibody production was outsourced to Bethyl Laboratories, Inc., Montgomery, Texas USA (bethyl.com). According to Bethyl Laboratories, rabbits were immunized, sera were collected. Sequential bleeds were screened by ELISA against the peptide to determine titer. Rabbits were euthanized according to ethical use protocols held by Bethyl Laboratories. Antibodies were affinity purified, concentrated, and stored at -20oC upon arrival.
Protein isolation and detection by immunoblot. Wildtype, heterozygotes (Col11a1a+/−) and homozygotes (Col11a1a−/−) embryos were used for protein isolation and detection by immunoblot. Each experimental group contained 20 embryos for protein extraction. Embryos were dechorionated using 1 mg/mL pronase at room temperature then rinsed in Ringer’s solution. Embryos were treated with ethylenediamine tetraacetic acid and protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific) in Ringer’s and passed through a glass pipette to remove the yolk.
Samples were processed as previously described [27] with modification. In brief, samples previously fixed in 4% paraformaldehyde (PFA) were incubated in 2% sodium dodecyl sulfate (SDS) at 95°C for 30 min and 60°C for 2 hours to reverse PFA fixation. The embryos were pelleted by centrifugation for 20 min at 5000 x g and the supernatant was removed. De-yolked embryos were homogenized in SDS sample buffer with a microfuge pestle, boiled, and centrifuged to clarify the extract. Extracted proteins were separated on a 4–12% gradient bis-Tris gel and transferred to polyvinylidene difluoride membranes and blocked for 60 min at room temperature. Membranes were rinsed with Tween-20 Tris buffered saline (TTBS) 3 x 5 min. Primary antibody was added to the membrane in TTBS with 5% bovine serum albumin and incubated overnight at 4oC with constant rocking. For peptide blocking experiments, the peptide was added to the primary antibody solution and agitated for 1 hour before adding to the membrane. The primary antibody solution was decanted, and unbound antibody was removed by rinsing the membrane with TTBS 3 x 5 min. Secondary antibody conjugated to horseradish peroxidase was added to the membrane and rocked at room temperature for one hour. The secondary antibody was decanted, and the membrane washed 3 x 5 min with TTBS. Enhanced chemiluminescence reagent was added to the membrane. Imaging and quantification of bands was performed on a FluorChem E Digital Darkroom. Band intensity was expressed as the mean plus and minus standard deviation, N = 3 independent replicates.
LC-MS/MS-based protein sequence analysis. LC-MS/MS analysis was performed using methods established previously [28] with modifications. Briefly, excised gel pieces were destained in 50% acetonitrile and 50mM NH4HCO3, followed by the treatment with dithiothreitol (10 mM) to reduce disulfide bonds and iodoacetamide (55 mM) for alkylation and then digested with proteomics grade trypsin (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 37oC. Peptides were extracted, dried under vacuum, and reconstituted in 5% acetonitrile and 0.1% formic acid. Tryptic peptides were analyzed on a Velos Pro Dual-Pressure Linear Ion Trap mass spectrometer equipped with a nano electrospray ionization source and coupled with an Easy-nLC II nano LC system (Thermo Fisher Scientific, Waltham, MA, USA).
Peptide spectral matching and protein identification were achieved by database search using Sequest HT algorithms in Proteome Discoverer 2.2 (Thermo Fisher Scientific). Raw spectrum data were searched against the UniProtKB/Swiss-Prot protein database for Zebrafish (downloaded from www.uniprot.org on September 8, 2020). Search parameters included: trypsin, maximum missed cleavage site of two, precursor mass tolerance of 1.5 Da, fragment mass tolerance of 0.8 Da, fixed modification of cysteine carbamidomethylation (+ 57.021 Da), and variable modification of oxidation/hydroxylation of methionine, proline, and lysine (+ 15.995Da). Decoy database search was performed to calculate false discovery rate (FDR). Proteins containing two or more peptides with FDR ≤ 0.01were considered positively identified.
Immunofluorescence. Wildtype, heterozygotes (Col11a1a+/−) and homozygotes (Col11a1a−/−) embryos were used for immunofluorescence analysis. Each experimental group contained 15 embryos. Zebrafish embryos were fixed in 4% PFA and embedded in paraffin prior to sectioning. Samples were cut into 10 micrometer sections. Sections were deparaffinized in xylenes and rehydrated in a graded alcohol series. Sections were rinsed with 0.1% Tween-20 in PBS and Triton X-100 at room temperature. Blocking was performed in 5% goat serum and 2% Tween-20 in PBS for one hour. Primary antibodies were diluted in blocking solution and samples were incubated at 4oC overnight. Secondary antibody was diluted 1:200 in blocking solution and applied to samples overnight in the dark. ProlongTM Gold antifade mountant with DAPI (Fisher Scientific) was used to mount the coverslip. Samples were imaged on a Zeiss LSM 510 Meta confocal microscope.