Rabbit anti calretinin

Pregnant females were euthanized by lethal intraperitoneal (i.p.) injection of pentobarbital (50 mg kg⁻¹), embryos were collected by caesarian cut and brains were dissected and fixed ON in cold 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer, pH 7.4. For postnatal brains, animals were deeply anaesthetized by i.p. injection of pentobarbital, transcardially perfused with 0.9% saline followed by cold 4% PFA and postfixed (ON) in cold 4% PFA. Brains were cut on a Vibratome (Leica, VT1000S) for IHC and for free-floating in situ hybridization. Sections were kept at 4 °C in 0.1 M phosphate buffer saline and were stained by IHC as described31 (link) with the following primary antibodies rabbit anti-GFP (1:500; Millipore), goat anti-GFP (1:1,000; Chemicon), mouse anti-human ovalbumin upstream promoter transcription factor 2 (COUP-TFII; 1:500; Perseus proteomics), goat anti-SP8 (1:50; Santa-Cruz), rabbit anti-NKX2.1 (1:100; Santa-Cruz), mouse anti-PV (1:1,000; Swant), rat anti- SST (1:500; Millipore), mouse anti-Reelin (1:500; Abcam), rabbit anti-VIP (1:500; Abcam), rabbit anti-NPY (1:1,000; Immunostar), rabbit anti-Calretinin (1:1,000; Swant). Secondary goat or donkey Alexa-488, -568 and -647 antibodies (Molecular Probes, Invitrogen) raised against the appropriate species were used at a dilution of 1:500–1,000 and sections were counterstained with Hoechst 33258 (1:10,000).

Mice were anaesthetized with an overdose of sodium pentobarbital and transcardially perfused with saline, followed by 4% paraformaldehyde (PFA). Brains were post-fixed for 2 h at 4°C. Brains were sectioned on a sliding microtome or a vibratome at 60 μm. All primary and secondary antibodies were diluted in PBS containing 0.25% Triton X-100 and 2% BSA. The following antibodies were used rabbit anti-Calretinin (1:1000, Swant), chicken anti-GFP (1:3000, Aves Lab), goat anti-Prox1 (1:300, R&D systems), mouse anti-reelin (CR-50, 1:300, MBL international) and rat anti-RFP (1:500, ChromoTek). We used Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch Labs and Molecular Probes).

Four days (96 h) after rhodamine injection, deeply anesthetized animals (sodium pentobarbital, 100 mg/kg, i.p.) were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in phosphate buffer 0.1 M, pH 7.4. Their brains were then dissected and cryoprotected in 30% sucrose prepared in phosphate buffered saline (PBS). Then, 40-µm-thick brainstem coronal sections were cut using a cryostat and collected in glycerol-PBS (1:1) for storage at −20 °C.
For the study of the effect of partial deafferentation and BDNF administration on axon terminal sprouting from abducens internuclear neurons, we used immunofluorescence against calretinin. Briefly, sections were rinsed, blocked with 7% normal goat serum (NGS) in PBS with 0.1% Triton X-100 (PBS-T), and incubated in the primary antibody (rabbit anti-calretinin, 1:5000; Swant, Burgdorf, Switzerland) at room temperature for 12 h. Sections were then washed and incubated for 2 h in the secondary antibody solution (goat anti-rabbit IgG coupled to Cy5, 1:200 in PBS-T; Jackson Immunoresearch, West Grove, PA, USA). After several washes in PBS, sections were mounted on glass slides and coverslipped with DakoCytomation (Dako, Glostrup, Denmark).

For immunostaining we followed protocols published in Ferran et al. (2015 (link)) and Garcia-Calero and Scharff (2013 (link)). The primary antibodies used in this study were: mouse anti-RC2 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-TH (1:1000; Novus Biologicals), mouse anti-parvalbumin (1:2000; Sigma-Aldrich), rabbit anti-Calretinin (1:1500, SWANT). Some sections were counterstained with neutral red.

The mice were anesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde in PBS. The brain was removed and kept overnight in 4% paraformaldehyde, after which it was cut into 100-µm-thick coronal sections using a vibratome (Leica, VT1200S). Brain sections were processed for fluorescent immunohistochemistry^{82 (link)}. The sections were permeabilized with 1% Triton X-100 in 1X Tris-Buffered Saline (1X TBS: 50 mM Tris, 150 mM NaCl, pH 7.4 adjusted with 1 M HCl) and incubated in blocking solution (5% normal donkey serum (Jackson, 017-000-12) and 0.4% Triton X-100 in 1X TBS). Rabbit anti-calretinin (1:1000; Swant, 7697) and rabbit anti-GluR2/3 (1:100; Millipore, AB1506) were used as primary antibodies (4 °C overnight incubation) for DRD2-Cre mice, while rabbit anti-calbindin-D28k (1:1000; Swant, CB38) was used for POMC-Cre mice. Donkey anti-rabbit Alexa Fluor 594 (1:500–1000; Jackson ImmunoResearch, 711-585-15) was used as a secondary antibody (3-h incubation at room temperature). Slices were washed in TBS and mounted using Vectashield mounting medium with DAPI (Sigma-Aldrich). Images of DAPI, GFP and Alexa 594 fluorescence were acquired separately with a confocal microscope (Nikon A1).

Tissue cultures were fixed in 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) for 4 h. After several washes with 50 mM Tris–buffered saline (TBS; pH 7.4), cultures were re-sliced into 30 μm sections on a vibratome (VT 1000S, Leica). For cutting on a cryostat cultures were fixed in 4% PFA in 0.1 M PBS (pH 7.4) and 4% sucrose for 1 h at room temperature (RT), followed by 2% PFA and 30% sucrose in PBS overnight at 4°C. After several washes with 50 mM TBS (pH 7.4), cultures were re-sliced into 30 μm sections on a cryostat (CM3050 S, Leica). Free-floating sections were washed several times in 50 mM TBS containing 0.1% Triton X-100, incubated in a blocking buffer (0.5% Triton X-100, 5% bovine serum albumin (BSA) in 50 mM TBS) for 30 min at RT and subsequently incubated with the appropriate primary antibody (diluted in 0.1% Triton X-100, 1% BSA in 0.05 M TBS) for 2 days at RT. Rabbit anti-calretinin (1:1000, Swant) and mouse anti-NeuN (1:1000, Chemicon) were used as primary antibodies. After several washing steps, sections were incubated with the appropriate secondary Alexa-conjugated antibodies (1:2000, Invitrogen) for 4 h at RT, counterstained with DRAQ5 (1:10,000, Thermo Fisher Scientific) or Hoechst (1:50,000, Sigma-Aldrich) to visualize nuclei, and mounted in Fluorescence Mounting Medium (Dako, Agilent Technologies).