Paraffin sections were deparaffinized, rehydrated, and subjected to citrate-based antigen retrieval. In other cases, 50-μm vibratome sections were used for staining. Primary antibodies against the following were used as follows: GFAP (DAKO; BD Biosciences), Ki67 (Novocastra), nestin (BD Biosciences), Olig2 (Chemicon), β-galactosidase (ICN), Doublecortin (Santa Cruz), NeuN (Chemicon), MBP (Sternberger), GFP (Chemicon), PDGFRα (Santa Cruz), NG2 (Millipore), CNPase (Millipore), APC (Calbiochem), BrdU (Abcam), Map2 (Sigma), Parvalbumin (Swant), Calretinin (Millipore), vGlut2 (Millipore), GABAARα (Millipore), Synapsin (Millipore), Sox2 (Abcam), γ-H2Ax (Cell Signaling), p21 (BD), p27 (Cell Signaling), p16 (Abcam), Lc3b (Novus Biologicals), p62 (MBL), pAKT (Cell Signaling), pErk (Cell Signaling) and pS6 (Cell Signaling). We used both immunofluorescence staining using Cy2, Cy3, or Cy5 (Jackson Labs) and biotin-streptavidin-Alexa Fluor-conjugated secondary antibodies (Molecular Probes), as well as horseradish peroxidase-based Vectastain ABC Kit (Vector Lab). The same antibodies were used for immunoblotting for γ-H2Ax, p21, p27, p16, Lc3b and p62. All antibodies were verified using appropriate positive and negative controls. Detailed information on all antibodies used are in the Supplementary Information section .
Parvalbumin
Manufactured by Swant
Sourced in Switzerland, United States
Parvalbumin is a calcium-binding protein found in various cell types, including neurons and muscle cells. It plays a role in regulating intracellular calcium levels and is commonly used as a marker for specific cell populations in research and diagnostic applications.
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Lab products found in correlation
Calretinin, by Merck Group (2 mentions)
Gad67, by Merck Group (2 mentions)
Parvalbumin, by Merck Group (2 mentions)
Cm3050, by Leica (2 mentions)
Calretinin, by Swant (2 mentions)
Calbindin, by Swant (2 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (2 mentions)
Bromodeoxyuridine (brdu), by Abcam (1 mentions)
Olig2, by Merck Group (1 mentions)
Vglut2, by Merck Group (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Pdgfrα, by Santa Cruz Biotechnology (1 mentions)
Doublecortin, by Santa Cruz Biotechnology (1 mentions)
Cnpase, by Merck Group (1 mentions)
Synapsin, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
10 protocols using parvalbumin
1
Immunohistochemical Profiling of Neural Markers
2
Immunohistochemical Analysis of Brain Markers
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Tissue stains were performed with 1:500 primary antibody and 1:500 Alexa Fluor 594 secondary antibody (Thermo Fisher Scientific), both overnight at 4°C. Primary antibodies were: MEF2D (Millipore #AB2263), Parvalbumin (Swant #PVG213), GFAP (Cell Signaling #3670), GAD67 (Millipore #MAB5406). The ApopTag TUNEL stain kit (Millipore) was used.
3
Immunolabeling of Nrp2 Mouse Brains
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Nrp2+/+ (wild-type), Nrp2+/− (heterozygote) and Nrp2−/− (homozygote) mice aged 3–6 months (average age of all genotype groups: 3.7 ± 0.95 months, four females and five males), when mice are considered to be in the mature adult stage [34 ], were subjected to transcardial perfusion with 4% paraformaldehyde (PFA). Brains were dissected, postfixed for 2 h in 4% PFA and then imbedded in OCT medium. Brains were sectioned on a cryostat at 20 μm thickness at 200 μm intervals through the anterior to the posterior extent of the hippocampus and cortex along the coronal plane. Sections were processed for immunocytochemistry as previously described [35 (link)], with the following modification. Coverslips were mounted on microscope slides using Mowiol (cat. No. 81381 Aldrich), plus 10% p-Phenylenediamine (PPD, cat No. 78460) anti-fade mounting media. Primary antibodies used were: mouse monoclonal anti-Parvalbumin (1.5:500, Swant, PV235); mouse monoclonal anti-Parvalbumin (1:750, Sigma, P3088); rabbit polyclonal anti-neuropeptide Y (1:1000, Abcam, ab30914); rat monoclonal anti-Somatostatin (1:150, Millipore, MAB354) and for visualization, AlexaFluor 488 or Cy5 (1:500, Jackson Immuno Research Laboratories); AlexaFluor 546 (1:500 Invitrogen).
4
DRG Cryosectioning and Immunohistochemistry
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L4-L5 DRG tissue were extracted, fixed in 4% paraformaldehyde dissolved in PBS, cryoprotected in 30% sucrose, and frozen in OCT (Tissue-Tek). Ten-μm thick cryosections were blocked with 1% bovine serum albumin (Sigma-Aldrich)/0.1%Triton X-100 in 0.1 M phosphate buffered saline (PBS) and then incubated with primary antibodies overnight at 4°C. After 3 washes in PBS for 10 minutes each, sections were incubated with secondary antibodies for 1 hour at room temperature, washed 3 times in PBS (10 minutes each) and mounted using Dako mounting medium (S3023). The following primary antibodies were used: Isolectin B4 (IB4) DyLight 594-conjugated (Vector laboratories FL-1207); CGRP (Millipore; PC205L), DDX3X (santa cruz; sc-365768); NF200 (Abcam; ab4680); Parvalbumin (Swant; PV-25). Secondary antibodies used: Alexa Fluor 555 anti-rabbit (Invitrogen; A32794), Alexa Fluor 555 anti-mouse (Invitrogen; A32773); Alex Fluor 488 anti-rabbit (Invitrogen; A21206); beta-tublin-488 (BD Pharmingen; 560338).
5
Immunohistochemical Profiling of Retinal Cells
6
Immunocytochemical Analysis of Neuronal Markers
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Immunocytochemistry was as described previously (Larsen et al., 2016 (link)). Primary antibodies consisted of monoclonal COUPTFII (1:500; Perseus Proteomics PP-H7147-00, Tokyo, Japan), polyclonal panDLX (1:1000; gift from Dr. Yury Morozov, Yale University, New Haven, CT), monoclonal βIII-Tubulin (1:5,000; Sigma T8660), polyclonal Calretinin (CALB2; 1:1000; Swant 7697, Switzerland), monoclonal Parvalbumin (1:5000; Swant PV235), and polyclonal MAP2 (1:2000; BioLegend 822501, San Diego, CA). Secondary antibodies used were: anti-mouse CF555, anti-rabbit CF488 (both at 1:2000; Sigma SAB4600060 and SAB4600036), and anti-chicken CF647 (1:500; Sigma SAB4600179).
7
Immunohistochemistry for Interneuron Markers
8
Immunostaining of Brain Tissue Markers
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Brain tissue preparation and immunohistochemistry were performed as described (Bormuth et al.28 (link)). Primary antibodies were directed against Ank3 (1:50, mouse IgG, Santa Cruz Biotechnology), Calretinin (Calb2; 1:1000, polyclonal rabbit; Millipore), HCN1 (1:200, polyclonal rabbit; Biomol), GABA Aα1 receptor (1:500, polyclonal rabbit, Millipore), GABA Aα6 receptor (1:500, polyclonal rabbit, Millipore), GAD67 (1:1000, mouse IgG, Millipore), GAD65 (1:1000, polyclonal rabbit, Millipore), GFAP (1:200 mouse IgG, Chemicon), MAP2 (1:800, mouse IgG, Millipore), NFH (1:200, polyclonal rabbit, Sigma), Parvalbumin (1:1000, mouse IgG, Sigma), Parvalbumin (1:000, polyclonal rabbit, Swant), Pax2 (1:250, polyclonal rabbit, Zymed), PCNA (1:300, polyclonal rabbit, Abcam), PSD95 (1:400, mouse IgG, Affinity Bioreagents), S100beta (1:200 monoclonal rabbit, Abcam), VGLUT1 (1:5000, polyclonal guinea pig, Millipore). Detection was performed with secondary antibodies conjugated to Alexa Fluor 488, 555 and 633 (1:1000, Thermo Fisher Scientific) and biotinylated secondary antibodies followed by diaminobenzidine (DAB; LSAB2 Kit, Dako; Vectastain Kit, Vector Laboratories).
9
Immunohistochemical Analysis of Brain Sections
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Brain coronal sections were washed three times in phosphate buffered saline (PBS) and quenched with 3% H2O2 for 20 min. Afterwards, sections were blocked with 5% normal donkey serum (NDS) in PBS supplemented with 0.25% Triton X-100 (PBS-T) for one hour. Primary antibodies were incubated overnight at room temperature or at 4 °C in 3% NDS PBS-T. The following primary antibodies were used: TH (rabbit cat# AB152, EMD Millipore, Temecula, CA, USA, 1:1000), Parvalbumin (goat cat# PVG213, Swant, Marly, Fribourg, Switzerland, 1:3000), Iba1 (rabbit cat# 019-19741, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan, 1:1000). After incubation with primary antibody, sections were incubated with corresponding biotinylated secondary antibodies (Jackson Immunoresearch, Baltimore, MA, USA, 1:400), in 3% NDS PBS-T, at room temperature for 90 min, and the signal was enhancement by using Vectorstain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA). Staining was revealed using chromogen 3,3-diaminobenzidine-tetrahydrochloride (Dabsafe, Saveen Werner AB, Limhamn, Sweden) and 3% H2O2. Sections were dehydrated in consecutive higher concentrations of ethanol, followed by xylene and mounted using Pertex (Histolab AB, Gothenburg, Sweden).
10
Immunohistochemistry for Interneuron Markers
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The fixed tissue was immersed in 20% and 30% sucrose solutions for two overnights, frozen and sectioned into 30 μm sagittal slices (Cryostat, Leica CM3050S). Following work in zebra finches47 (link), the slices were stained for calcium binding interneuron markers Calbindin (1:4000, SWANT), Calretinin (1:15000, SWANT), and Parvalbumin (1:1000, SWANT) by overnight incubation with the primary antibody at 4°C and with a secondary antibody (coupled to Alexa Fluor 647) for 2 hours at room temperature. Slices were mounted on microscope slides, and sealed with cover slips and nail polish. A confocal microscope (Nikon C2si) was used to image GCaMP6f and the interneuron markers in 3μm-thick sections through the tissue. The images were inspected for co-stained cells (e.g. SI videos 1 –7 ). The results ruled out any co-expression of GCaMP and Calbindin or Calretinin. We found 2 cells expressing Parvalbumin and GCaMP (SI video 5 shows one example, <0.5% of PV stained cells, <0.01% of GCaMP expressing cells), possibly replicating previous observation of PV expression in HVC projection neurons47 (link).
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