Calretinin

For in situ hybridization analysis, cryostat sections were hybridized using digoxigenin-labeled probes [45 (link)] directed against mouse TrkA or TrkB, or rat TrkC (gift from L. F. Parada). Antibodies used in this study were as follows: rabbit anti-Er81 [14 (link)], rabbit anti-Pea3 [14 (link)], rabbit anti-PV [14 (link)], rabbit anti-eGFP (Molecular Probes, Eugene, Oregon, United States), rabbit anti-Calbindin, rabbit anti-Calretinin (Swant, Bellinzona, Switzerland), rabbit anti-CGRP (Chemicon, Temecula, California, United States), rabbit anti-vGlut1 (Synaptic Systems, Goettingen, Germany), rabbit anti-Brn3a (gift from E. Turner), rabbit anti-TrkA and -p75 (gift from L. F. Reichardt), rabbit anti-Runx3 (Kramer and Arber, unpublished reagent), rabbit anti-Rhodamine (Molecular Probes), mouse anti-neurofilament (American Type Culture Collection, Manassas, Virginia, United States), sheep anti-eGFP (Biogenesis, Poole, United Kingdom), goat anti-LacZ [14 (link)], goat anti-TrkC (gift from L. F. Reichardt), and guinea pig anti-Isl1 [14 (link)]. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to detect apoptotic cells in E13.5 DRG on cryostat sections was performed as described by the manufacturer (Roche, Basel, Switzerland). Quantitative analysis of TUNEL⁺ DRG cells was performed essentially as described [27 (link)]. BrdU pulse-chase experiments and LacZ wholemount stainings were performed as previously described [46 (link)]. For anterograde tracing experiments to visualize projections of sensory neurons, rhodamine-conjugated dextran (Molecular Probes) was injected into single lumbar (L3) DRG at E13.5 or applied to whole lumbar dorsal roots (L3) at postnatal day (P) 5 using glass capillaries. After injection, animals were incubated for 2–3 h (E13.5) or overnight (P5). Cryostat sections were processed for immunohistochemistry as described [14 (link)] using fluorophore-conjugated secondary antibodies (1:1,000, Molecular Probes). Images were collected on an Olympus (Tokyo, Japan) confocal microscope. Images from in situ hybridization experiments were collected with an RT-SPOT camera (Diagnostic Instruments, Sterling Heights, Michigan, United States), and Corel (Eden Prairie, Minnesota, United States) Photo Paint 10.0 was used for digital processing of images.

Animals were anesthetized with a lethal dose of ketamine (40 mg/100 g body weight, i.p.) and xylazine (2 mg/100 g body weight, i.p.). When a deep anesthetic state marked by a complete loss of the flexor reflex at all limbs was reached, animals were perfused transcardially with 20 mL of phosphate buffered saline (0.1 M PBS, pH 7.4) supplemented with 0.1 % heparin followed by 200 mL of 4 % PFA (in 0.05 M PBS, pH 7.4). The brains were postfixed in the skull with 4 % PFA (in 0.05 M PBS, pH 7.4) at 4 °C for at least 7 days before removal to best preserve the brain shape.
Brains were cryo-protected in 22.5 % sucrose in PBS (0.05 M, pH 7.4) overnight and cut in a cryostat (LEICA CM 3050S) into four series of 40 µm thick frontal sections. The sections were directly mounted on gelatine-coated slides and dried overnight. Alternating section series were stained on-slide either for cells (Nissl) or for myelin (Gallyas 1979 (link)). The brains additionally processed for chemo- and immunoarchitecture were stained for cytochrome oxidase, acetylcholine-esterase (AChE), NADPH-diaphorase, calcium-binding proteins (parvalbumin, calbindin and calretinin) and neurofilament protein (SMI-32) in various combinations. Sections were imaged with a virtual slide microscope (VS120 S1, Olympus BX61VST, Olympus-Deutschland, Hamburg, Germany) at 10× magnification using the proprietary software dotSlide^® (Olympus).

C5–C7 spinal segments or musculocutaneous nerves were cut into 15-µm-thick frozen sections for immunostaining. The blocking buffer was composed of 5% goat serum and 3% bovine serum albumin diluted in 0.1 M phosphate buffer saline (PBS). Signal was detected with Alexa fluor 546 or 488 coupled secondary antibodies (1:1000, Invitrogen). Primary antibodies were: goat anti- choline acetyltransferase (ChAT, 1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Calretinin (1:300, ab702, Abcam), mouse anti-Parvalbumin (1:1000, Mab1572, Millipore), rabbit anti-CAMKII (1:500, ab104224, Abcam), rabbit anti-vesicular GABA transporter (VGAT; 1:800, NO131013, Synaptic Systems), mouse anti-vesicular glutamate transporter 1 (vGlut1; 1:1000, Mab5502, Millipore), rat anti-major histocompatibility complex 1 (MHC1; 1:300, sc-59199, Santa Cruz), rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, AB7260, Abcam), rabbit anti-Iba1(1:1000, 019–19,741, Wako), and rabbit anti-Oligo2 (1:500, ab9610, Merck Millipore).
On day 50 after BPA, the biceps were collected and 7-µm horizontal sections were prepared with a sliding microtome (Leica, Germany) and double stained with rabbit anti-NF200 (1:500, n4142, Sigma) and α-BT (1:1000, Molecular probes, USA) to visualize neuromuscular junctions (NMJs).

Myh10 en face apical endfoot images, Myh9 3D reconstructions, and Myh9 LHX6/Laminin images were captured using an Andor Dragonfly Spinning Disk Confocal plus with 40X and 63X objectives. All other images were captured using a Zeiss Axio Observer Z.1 with Apotome for optical sectioning. 20X, 40X, and 63X objectives were used. For each experiment, 3 to 4 sections per embryo were imaged. Identical exposures, apotome phase images, and Z intervals were used. Cell counting was performed manually (FIJI cell counter) or automatically (QuPath). For QuPath quantification, the following parameters were used: requested pixel size = 0.1 μm, background radius = 5 μm, minimum area = 10 μm², maximum area = 200 μm², cell expansion = 2 μm, include cell nucleus and smooth boundaries were unselected. For quantification of cells touching the BM, cells colocalizing with the BM label (Laminin or Collagen) were counted manually. Cortical, SOX2, MZ, and Calretinin thickness measurements were taken using the line tool in ImageJ.

In utero BioID brains were fixed prior to microdissection for immunofluorescence analysis. Briefly, embryonic brains were fixed overnight in 4% formaldehyde/PBS, rinsed with PBS, incubated in 30% sucrose/PBS overnight, and then embedded in NEG-50 (Thermo Scientific, 6052). Unless otherwise specified, 20 μm sections were used for all staining. For antibody staining, sections were thawed, rinsed in PBS, permeabilized with 0.25% Triton X-100/PBS, blocked with 5% NGS/PBS, incubated with primary antibody in block buffer overnight at 4°C or room temperature for 2 hours, rinsed 3 times with PBS, incubated with species-specific secondary antibody (Alexa Fluor conjugated, Thermo 1:500) and DAPI in block buffer for 30 minutes or 2 hours at room temperature (LHX6 and p73), rinsed 3 times with PBS, and mounted with Vectashield Anti-Fade Mounting Medium (Vector Labs, H-1000). The following primary antibodies were used: Rabbit: anti-HA (Santa Cruz, sc-805, 1:250), anti-MYH9 (Biolegend, 909802 1:1,000), anti-MYH10 (Biolegend, 909902 1:1,000), anti-p73 (Cell Signaling, 14620S 1:250), anti-Calretinin (Swant, CR7697 1:1,000), anti-ISG15 (Thermo, 703132 1:100), anti-FERMT3 (Proteintech, 18131-1-AP 1:100), anti-TNS3 (Invitrogen, PA5-63112 1:75), anti-CC3 (Cell Signaling, 9661 1:400), anti-Ki67 (Cell Signaling, 12202 1:250), anti-Laminin (Millipore, AB2034 1:500), Anti-Collagen I (Invitrogen, PA5-95137 1:500), Anti-PSEM2 (ProteinTech, 12937-2-AP, 1:100) Mouse: anti-P150 Dynactin (BD Biosciences, 610473, 1:200), anti-LHX6 (Santa Cruz, sc-271 433, 1:500), anti-Reelin (Millipore, mab5364 1:100) Rat: anti-SOX2 (Thermo, 14-9811-82 1:500) Other: Streptavidin-Alexa Fluor 594 conjugate (Thermo Scientific, S11227, 1:500).