Computational protocol: The control of alternative splicing by SRSF1 in myelinated afferents contributes to the development of neuropathic pain

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Protocol publication

[…] Rats were terminally anesthetized with sodium pentobarbital overdose (i.p. 60 mg/kg) and were perfused transcardially with saline followed by 4% paraformaldehyde. The L3-4 segments of the lumbar enlargement, containing the central terminals of saphenous nerve neurons (), and L3-L4 dorsal root ganglia were removed, post fixed in 4% paraformaldehyde for 2 h and cryoprotected in 30% sucrose for 12 h. Tissue was stored in OCT embedding medium at − 80 °C until processing. A cryostat was used to cut spinal cord (20 μm) and dorsal root ganglia (8 μm) sections that were thaw mounted onto electrostatic glass slides. Slides were washed in phosphate buffered saline (PBS) solution 3 times for 5 min per incubation, and incubated in PBS 0.2% Triton X-100 for 5 min. Sections were blocked (5% bovine serum albumin, 10% fetal bovine serum, 0.2% Triton X-100 in PBS) for 2 h at room temperature, and then incubated in primary antibodies diluted in blocking solution overnight at 4 °C. Sections were washed three times in PBS washes and incubated for 2 h in secondary antibody (e.g. biotinylated or alexafluor-conjugated; 0.2% Triton X-100 in PBS). For the third stage (i.e. streptavidin-alexfluor conjugate), incubations and washes were as described for the secondary antibody. Slides were washed in PBS 3 times prior to coverslipping in Vectorshield (H1000 or H1200 containing DAPI for nuclear staining, Vector Laboratories). Images were acquired on either Nikon Eclipse E400 and a DN100 camera or Leica TCS SPE confocal microscope using Leica application suite (Tumor and Vascular Biology Laboratories' imaging suite UoN).Primary antibodies used were as previously reported (, ): anti-ATF3 (rabbit polyclonal; 2 μg/ml: Santa Cruz), anti-c-fos (rabbit polyclonal; 2 μg/ml: Santa Cruz), anti-SRSF1 (goal polyclonal; 2 μg/ml; sc-10,255 Santa Cruz), anti-vGLUT1 (rabbit polyclonal, 60 pg/ml, Synaptic Systems), anti-NF200 (mouse monoclonal; 1.4 μg/ml; N0142 Sigma-Aldrich), anti-NeuN (mouse monoclonal, 1 in 100, Millipore). Use of anti-VEGF-A and SRSF1 antibodies for both immunolocalization and immunoblotting has been previously reported (, ). Secondary antibodies (1 in 1000 dilution and from Invitrogen unless stated): Alexafluor 488 goat anti-mouse, Alexafluor 488 chicken anti-goat, Alexafluor 555 donkey anti-goat, Alexafluor 555 donkey anti-rabbit; biotinylated anti-rabbit (Stratech Scientific), Extravidin CY3 (Sigma-Aldrich). Dorsal root ganglia neuronal cell counts were performed using ImageJ analysis to measure neuronal area (μm2) (). The saphenous nerve is approximately equally derived from lumbar DRGs 3 and 4 in rat and human (, , ); the mean number of neurons per section was quantified from 10 non-sequential random L4 DRG sections per animal. Data are presented as the mean number of neurons per section and the experimental unit is the animal. The number of activated SRSF1-positive neurons (defined as those showing nuclear localization of SRSF1) was calculated as a percentage of total neurons as designated by size (small < 600 μm2, medium 600 μm2–120 0 μm2, large > 1200 μm2) (). The total number of DRG neurons quantified was ~ 5000 (100 neurons per section, 10 per animal, 3 per group). Determination of SRSF1 spinal cord expression/localization was determined from 5 non-sequential random spinal cord sections per animal using Image J analysis. Images were converted to an 8-bit/grayscale image then thresholding was applied across all acquired images to determine the area of positive staining. Areas of positive staining were then quantified across all sections and groups. Colocalization was determined via coloc2 plugin in ImageJ. Controls for VEGF-A and SRSF1 immunofluorescence consisted of incubation with only secondary antibody (‘no primary’ control) or substitution of the primary antibody with a species matched IgG. […]

Pipeline specifications

Software tools ImageJ, Coloc
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Cranial Nerve Injuries
Chemicals Formaldehyde