Immunohistochemistry was performed as previously described (Lin et al., 2018 (link)). Mice were transcardially perfused with 4% PFA in PBS and the spinal cord were stored in 1% PFA at 4°C overnight. Samples were transferred to 20% and 30% sucrose solution for dehydration at 4°C and embedded with O.C.T medium (Fisher Healthcare). Frozen blocks were cut with cryostat (Thermofisher, USA) or vibratome (Leica, VT1000 S) to prepare 20-60 μm-thick transverse slices. For somatodendritic analysis of ErbB4+ cells, 400-μm thick slices were prepared. Slices were washed with PBS and blocked with PBS containing 0.5% Triton-100 and 10% donkey serum for 1 hr at RT, before overnight incubation of primary antibodies at 4°C. After wash, slices were incubated in AlexaFluor secondary antibodies (Jackson ImmunoResearch) for 2 hr at RT. Slices were washed, mounted onto slides and covered with SlowFade Diamond Antifade Mountant (catalog #S36972; Thermo Fisher). The following primary antibodies were used for immunostaining: mouse anti-c-Fos (#271243, 1:500; Santa Cruz), rabbit anti-c-Fos (#ABE457, Millipore), chicken anti-GFP (#1020, 1:1,000, Aves), anti-RFP (#600-401-379-RTU, 1:500, Rockland), and rabbit anti-NeuN (#R-3770-100, 1:500, Biosensis). Alexa-647 conjugated streptavidin (#S21374, 1:200, Life technologies) was used for visualization of biocytin.
Mouse anti c fos
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-c-Fos is an antibody product that specifically binds to the c-Fos protein. c-Fos is an immediate early gene that serves as a transcription factor, playing a role in cellular processes. This antibody can be used to detect and study the expression of c-Fos in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rabbit anti substance p, by GeneTex (2 mentions)
Alexa fluor secondary antibody, by Jackson ImmunoResearch (1 mentions)
Vt1000, by Leica (1 mentions)
Anti rfp, by Rockland Immunochemicals (1 mentions)
Slowfade diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa 647 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti c fos, by Merck Group (1 mentions)
Oct medium, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Microm hm 560 cryostat, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Goat anti rabbit igg, by Boster Bio (1 mentions)
Rabbit anti tgf β1, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Boster Bio (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Vt1200, by Leica (1 mentions)
Zen 2012 imaging software, by Zeiss (1 mentions)
6 protocols using mouse anti c fos
1
Immunohistochemistry for Spinal Cord Analysis
2
Protein Expression Analysis Protocol
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Tissue specimens were lysed in a lysis buffer (100 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol) containing protease inhibitors. Total protein concentration was assessed via the BCA method. The total protein was kept at −80°C awaiting subsequent analysis. Equal amounts of proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology and transferred to PVDF (polyvinylidene fluoride) membrane. 5% diluted skimmed milk powder was used as a sealant and imprinted in Tris-Buffer salt water for 3 h at a time. Rabbit anti-substance P (dilution: 1:500, GeneTex, Inc., United States) and mouse anti-c-fos (dilution: 1:200, Santa Cruz Biotechnology, United States) monoclonal antibodies were blotted at 4°C for overnight incubation. After washing, the membrane was incubated with conjugated goat anti-mouse IgG (1:8000) or goat anti-rabbit IgG (1:5000) for 2h at room temperature. Next, the membrane was washed with buffer. Immune response bands were examined using the ChemiDoc XRS + Bio-Rad imaging system. Image J software (National Institutes of Health) was applied to determine the quantitative density of the membrane, with beta-actin (dilution: 1:1000, Santa Cruz Biotechnology, United States) as the control.
3
Circadian Rhythm Analysis in Mouse SCN
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Mice were perfused with PBS/4% PFA at 4h intervals over 24h of a 12:12LD cycle. Brains were incubated for 90 min in PBS/4% PFA at RT then in 30% sucrose solution in PBS overnight at 4°C, followed by freezing. 40μm coronal slices including the SCN were cut on a Thermo Scientific Microm HM 560 Cryostat. Free-floating SCN slices were washed 6x10 minutes in 10XPBS; incubated 1h in blocking solution (0.1M PBS, 2.5% NGS, 0.3% Triton-X, 0.005% sodium azide) at RT; incubated for 48h at 4°C in blocking solution plus mouse anti-c-Fos (1:50; Santa Cruz Biotechnologies), rabbit anti-VIP (1:600; Peninsula Laboratories) and/or chicken anti-GFP (1:1000); washed 6x in 10xPBS; incubated with 2° antibodies for 1h at RT in the dark; 6x10 min washes in 10xPBS with DAPI added to the last wash (1:1000). Slices were mounted using Vectashield (Vector Laboratories Inc.) then a Z stack was imaged on a Zeiss LSM 710 confocal microscope at 20x magnification. This image was converted into a Z-projection in ImageJ. The SCN was identified by DAPI staining, and a threshold was set to eliminate non-specific background outside the SCN. The option Analyze Particles was then used to quantify the number of cFOS positive cells within the SCN.
4
Western Blot Analysis of Biochemical Markers
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The specimens were lysed with lysis buffer (100 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol) containing protease inhibitors. The BCA method was used to determine the total protein concentration. Lysates with equal amounts of protein were resolved on SDS-PAGE, and then transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk in TBST buffer (100 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4 and 0.1% Tween-20) at 4°C overnight. The membranes were washed with TBST buffer and then incubated with monoclonal antibodies against rabbit anti-substance P(dilution: 1∶500, GeneTex, Inc., USA), mouse anti-c-fos (dilution: 1∶200, Santa Cruz Biotechnology, USA), mouse anti-NGF (dilution: 1∶200, Santa Cruz Biotechnology, USA), rabbit anti- TGF-β1 (dilution: 1∶300, Santa Cruz Biotechnology, USA), mouse anti-collagen I (dilution: 1∶300, Santa Cruz Biotechnology, USA) and III (dilution: 1∶200, Boster, Wuhan, China) at room temperature for 1.5 h. After washing, the samples were incubated for 2 hr at room temperature with HRP-conjugated goat anti-mouse IgG (1∶8 000) or goat anti-rabbit IgG(1∶5000). The membrane was then washed with buffer and the image scanned with a GS800 Densitometer Scanner. Optical density data were analyzed using PD Quest 7.2.0 software. Beta-actin (dilution: 1∶1000, Santa Cruz Biotechnology, USA) was used as a control.
5
Western Blot Analysis of Substance P and c-Fos
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The specimens were lysed with lysis buffer (100 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, and 10% glycerol) containing protease inhibitors (Roche). The bicinchoninic acid assay was used to determine total protein concentration. Equal amounts of protein were resolved on a sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in TBST buffer for 1 hour. The membranes were washed 3 times with TBST buffer and then incubated with monoclonal antibodies against rabbit antisubstance P (dilution: 1:400, GeneTex, Inc.) and mouse anti-c-fos (dilution: 1:200, Santa Cruz Biotechnology) at room temperature for 1.5 hours. After an additional 3 washes, the samples were incubated for 2 hours at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000) or goat anti-rabbit IgG (1:1000). The membranes were then washed with TBST buffer, and the images were scanned with a GS800 Densitometer Scanner. The optical density data were analyzed using Image-Pro Plus software (Media Cybernetics). We used beta-actin (dilution: 1:1000, Santa Cruz Biotechnology) as a control.
6
Immunostaining of Coronal Brain Sections
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Coronal brain sections were cut using a vibrating microtome (Leica VT 1200S; Leica Biosystems, Sydney, Australia) into 40 μm serial sections collected in five series. Free-floating sections were permeabilized in 50% ethanol for 30 min at RT and washed in tris-phosphate-buffered saline (PBS). Sections were incubated in primary antibodies (diluted in 10% normal horse serum in tris-PBS) for SST2A (guinea pig anti-SST2A, 1:1,000, Biotrend, Cologne, Germany) and c-Fos (mouse anti-c-Fos, 1:500, Santa Cruz Biotechnology Inc., Dallas, Tex) for 48 h at 4°C. Sections were then incubated in fluorescent conjugated secondaries (Cy5-conjugated donkey anti-guinea pig IgG, 1:250 and 488-conjugated donkey anti-mouse IgG, 1:250 respectively, Jackson ImmunoResearch, West Grove, Pa) overnight at 4°C and then washed, mounted on glass slides using Dako mounting medium, and visualized using a light microscope (Zeiss, Axio Imager Z.2, Oberkochen, Germany). Images were acquired using ZEN 2012 imaging software (Zeiss, Germany). High-power images were taken using a confocal microscope (Leica TCS SP8, Nussloch, Germany) and acquired using Leica Application Software AF (LAS AF; Leica, Nussloch, Germany). All images were imported and analyzed using the ImageJ plugin Fiji.
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