mouse anti f310  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Functional assessment of a kcnb1 knock-out zebrafish to model KCNB1 -related neurodevelopmental and epileptic disorders

doi: 10.1101/2024.07.03.601913

Figure Lengend Snippet: A. Whole-mount images of 48 hpf-zebrafish obtained with a brightfield microscopy and treated with Phalloidin preventing actin depolymerization to identify general muscle fiber organization ( and 8A b ). Fish were then labelled with anti-F310 and anti-F59 markers to differentiate, respectively, fast and slow muscle fibers ( and 8A d ). Control fish ( kcnb1 +/+ ) and kcnb1 mutant models ( kcnb1 +/− and kcnb1 −/− ) display well organized muscle structures with typical parallel line pattern at 48 hpf (n = 4-8 ZF/genotype; 3D reconstruction; lateral view; magnification 20x; scale bar: 50 μm). B. Quantification of the anisotropy of fibrillar structures in raw images of Phalloidin, F310 and F59 labelling using the plugin FibrilTool from the software ImageJ . A Gaussian filter was applied on images and the anisotropy was analyzed reflecting the organization of fibers following the convention: 0 for no order and 1 for perfectly ordered (parallel fibrils) (anisotropy of fast muscles (F310): kcnb1 +/+ = 0.443 ± 0.02; kcnb1 +/− = 0.387 ± 0.06; kcnb1 −/− = 0.51 ± 0.02; +/+ vs mutants: p>0.5; n = 5-12 ZF/genotype/marker; Mann-Whitney test; ns: non-significant). Values represent the mean ± standard error of the mean (SEM). C. Plot profile illustrating the relative distance between fibers for each muscle fibers labelling (Phalloidin, F310 and F59). The results show similar profiles between control and both mutant conditions reflecting well organized muscle fibers (n = 4-8 ZF/genotype/marker). D. Primary motoneuron axons of controls and kcnb1 mutant zebrafish at 48 hpf were immunostained with anti-Znp1 marker (green) and acetylcholine receptors (AchR) were labelled with Bungarotoxin (BTX, red) (n = 4-8 ZF/genotype, magnification 20x, scale bar: 50 μm). At 48 hpf, to ef1α mRNA expression and kcnb1 +/+ fish are considered as the reference value (relative mutant fish ( kcnb1 +/− and kcnb1 −/− ) present similar motor axons length and branching as compared to WT fish associated with colocalization with AchR puncta identified along axons, quantified with Imaris software and represented by yellow spots, indicating functional synapses. E. Quantification of motor axon length of fish at 48 hpf. An average of eight motor axon length per fish was measured and a ratio was applied according to the spinal cord thickness. The total length quantification did not show any difference in motor axon size between mutant zebrafish models and the control condition ( kcnb1 +/+ = 2.38 ± 0.11; kcnb1 +/− = 2.43 ± 0.06; kcnb1 −/− = 2.55 ± 0.14; +/+ vs mutants: p>0.5; n = 7–9 ZF/condition; Mann-Whitney test; ns: non-significant). Values represent the mean ± standard error of the mean (SEM). F and G. Total RNAs were extracted from fish at 6 dpf and RT-PCR was performed to determine the transcript level of (F) acetylcholinesterase (AchE) and of (G) subunits α1, γ, ε (chrn) of AchR. Data are normalized to ef1α mRNA expression and the condition kcnb1 +/+ is considered as the reference value (relative fold change = 1). Both mutant conditions ( kcnb1 +/− and kcnb1 −/− ) presented significant decrease of AchE expression, reflecting a neurotransmission recycling defect at the NMJ level . However, the composition of AchR subunits is similar between conditions . N = 3; n = 30/sample; Mann- Whitney test; ns: non-significant; *p<0,05; **p<0,01. Values represent the mean ± standard error of the mean (SEM). chrn: cholinergic receptor nicotinic.

Article Snippet: Primary antibodies used were mouse anti-acetylated tubulin antibody (1:100, Sigma-Aldrich, USA, #T7451), mouse anti-3A10 antibody (1:100, DSHB, Iowa City, Iowa, USA, #3A10), mouse anti-F310 antibody (1:100, DSHB, USA, #F310), mouse anti-F59 antibody (1:20, DSHB, USA, #F59), mouse anti-Znp1 antibody (1:100, DSHB, USA, #znp1) and mouse anti-c-Fos antibody (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-166940).

Techniques: Microscopy, Control, Mutagenesis, Software, Muscles, Marker, MANN-WHITNEY, Expressing, Functional Assay, Reverse Transcription Polymerase Chain Reaction