Alexa fluor 488 secondary antibody

Erythrocytes were isolated from 3-dpf zebrafish embryos (wild-type and miR-144^Δ/Δ) by dissecting their tail using sapphire blade (WPI) and collecting cells in the bleeding buffer (1× PBS containing 2% PBS and 5 mM EDTA). Erythrocytes were washed twice with the bleeding buffer and spread on Superfrost Plus microscope slides using Shandon Cytospin2 Centrifuge at 35× g for 5 min and fixed for 20 min in ice-cold methanol. Erythrocytes were incubated for 30 min at room temperature in blocking buffer and stained directly on slides with anti-RNAP II Phosho-Ser2 (Abcam #5095) in the blocking buffer (1X PBS, bovine serum albumin (BSA) 2%, Tween 0.2%) overnight at 4 °C. The slides were then washed three times with 1× PBS, after what anti-rabbit Alexa Fluor 488 secondary antibodies (Jackson Immuno Research Laboratories, #711-545-152) in blocking buffer were added and cells were incubated for 1 h at room temperature. After that, DAPI was added to cells and incubated for 10 min at room temperature. After final washes, 1× PBS was mounted using Antifade Mounting Media (Vestashield) for fluorescence microscopy imaging. Images were captured using Zeiss Axio Observer Z1 microscope equipped with a digital camera (C10600/ORCA-R2 Hamamatsu Photonics).

Embryonic brains were fixed in 4% PFA over-night and sectioned. 10 μm Cryo-sections were pre-treated using the RNAscope Universal Pretreatment Kit (Advanced Cell Diagnostics, Cat#322380). RNA In Situ Hybridizations (ISH) were performed using the RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics, Cat#320850) according to manufacturer’s instructions. The target genes (mouse Lphn1-3 and Ten1-4) are also listed in the Key Resources Table. Following ISH, sections were immunostained using mouse anti-Pvim 1/300 (Abcam, Cat#ab20346) and rat anti-Ctip2 1/600 (Abcam, Cat#ab123449) in combination with the Alexa Fluor 488 secondary antibodies 1/200 (Jackson Immunoresearch). Images were acquired using a Leica TCS SP8 confocal laser scanning microscope and processed with ImageJ software.

Brain sections were permeabilized with 0.5% TritonX‐100 in phosphate‐buffered saline (PBS) for 30 min at room temperature, blocked with 5% bovine serum albumin in PBS, and incubated with anti‐HMOX2 primary antibody (1:300, abcam, ab90492, polyclonal, Rabbit) or anti‐SERPINA3 primary antibody (1:200, ABclonal, A2803, polyclonal, Rabbit) for 24 h at 4°C. The second day, the brain sections were washed with 0.1% TritonX‐100 in PBS for three times and incubated with Alexa Fluor 488 secondary antibodies (1:300, Jackson ImmunoResearch, 111‐545‐003, anti‐Rabbit) for 1 h at room temperature. Then, the brain sections were washed with 0.1% TritonX‐100 in PBS for three times and incubated with 4’,6‐diamidino‐2‐phenylindole (DAPI) for 10 min. Finally, brain sections were washed with 0.1% TritonX‐100 in PBS for three times and covered with 50% glycerin in PBS. Pictures were visualized by LSM710 (Zeiss Carl LSM 710, Germany).

Immunohistochemistry was conducted to verify inflammatory responses in L4 and L5 DRGs. Under urethane anesthesia (1.25 g/kg, i.p.), each rat was transcardially perfused with normal saline (0.9% NaCl), followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.4). DRGs were removed and immersed in 4% paraformaldehyde in 0.1 M PB for 24 h at 4 °C for post-fixation. The tissues were kept in 30% sucrose in phosphate buffer (PB, pH 7.4) at 4 °C. For immunostaining, embedded tissues were cryosectioned to 10 μm (HM 525, Thermo Scientific, Waltham, MA, US). The section slides were incubated overnight at 4 °C with primary antibodies against Nav1.7 (1:500; cell signaling technology, Danvers, MA, USA), washed with PBS, and incubated for 2 h at room temperature with Alexa Fluor 488 secondary antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA, USA). DAPI was used for counterstaining. Immunofluorescent sections were imaged by LSM700 confocal microscope (Zeiss, Oberkochen, Germany) using 10× and 40× PlanApo oil-immersion lenses. Briefly, 12 μm-thick confocal Z-stacks of the synaptic zone in ZI were captured. Three image stacks per rat (4/group) were used for analysis, and the number of cells with Nav1.7 were quantified.

For parvalbumin (PV) putative puncta detection, we employed a similar immunostaining procedure as described above. Neurog2^CreER-Tdt adult mice (age> P50) of either sex were used. Goat anti-PV primary antibody (1:1000, Swant, pvg-214, RRID:AB_10000345) and donkey anti-goat AlexaFluor488 secondary antibody (1:500, Jackson Immunoresearch, RRID:AB_2336933) were used. To compare the PV perisomatic innervation across CA1PNs with different birthdates, image stacks (0.06 × 0.06 × 0.410 µm) centered on the soma of TdTomato + cells (E12.5 CA1PNs: 88 cells; E14.5 CA1PNs: 106 cells; E16.5 CA1PNs: 44 cells) were acquired with a Zeiss LSM-800 microscope using a Plan-Apo 40 x/1.4 oil objective. The mean total thickness of the stacks was 15,85 µm, consistent with the mean soma diameter of pyramidal cells in adult CA1 (Ishizuka et al., 1990 (link)). Volume overlaps between PV+ boutons and Tdt⁺ somata were calculated using a custom-made MATLAB script. Values were normalized by the number of Z-steps to control for possible differences in soma size or experimental variability.