For whole-mount staining, the organoids were first fixed using 4% paraformaldehyde (Thermo Fisher) for 30 min at room temperature and then permeabilized using 0.1% TritonX-100 (Sigma-Aldrich) in Tris-buffered saline (TBS) for 15 min. After blocking in DAKO (Agilent) for 1 h, organoids were incubated with primary antibodies overnight at 4°C. Next day, after triple washing, organoids were incubated in secondary antibodies (ThermoFisher) along with 4’, 6-diamidino-2-phenylindole (DAPI) for 1 h at room temp. The following primary antibodies were used: mouse anti-vimentin (Abcam), rabbit anti-calcitonin gene related peptide (CGRP) (Millipore Sigma), rabbit anti-EpCAM (Abcam), rabbit anti-synaptophysin (Abcam), mouse anti-chromogranin A (Proteintech), NCAM1 (Cell Signaling Technology). Confocal imaging was performed using Zeiss LSM 880.
Rabbit anti synaptophysin
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-synaptophysin is a primary antibody that specifically binds to the synaptophysin protein, which is a membrane glycoprotein found in presynaptic vesicles of neurons. This antibody can be used for the detection and analysis of synaptophysin in various applications, such as immunohistochemistry and western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Triton x 100, by Merck Group (2 mentions)
Rabbit anti psd95, by Abcam (2 mentions)
Rabbit anti gs, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Syn per synaptic protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Rabbit anti epcam, by Abcam (1 mentions)
Ncam1, by Cell Signaling Technology (1 mentions)
Mouse anti vimentin, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Aqua poly mount mounting medium, by Polysciences (1 mentions)
Rabbit anti 5 ht, by Immunostar (1 mentions)
Mouse anti actub, by Merck Group (1 mentions)
Mouse anti mash1, by BD (1 mentions)
Rabbit anti dcx, by Cell Signaling Technology (1 mentions)
Rabbit anti sert, by Immunostar (1 mentions)
Rabbit anti aciii, by Santa Cruz Biotechnology (1 mentions)
15 protocols using rabbit anti synaptophysin
1
Whole-Mount Staining of Organoids
2
Immunohistochemical Analysis of Synaptic Markers
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After the behavioral study, mice were deeply anesthetized with isoflurane, perfused with PBS, and fixed with 4% PFA. Their brains were removed and fixed in 4% PFA for 24 h. Brains were cut from the superior colliculus to the optic chiasma and separated along the median sagittal plane. The left parts were prepared for Golgi staining, and the right parts were routinely dehydrated, waxed, embedded, and cut into 5 μm-thick sections. After dewaxing, hydration, high pressure antigen repair, and blocking, the sections were incubated at 4 °C overnight with the following primary antibodies: rabbit anti-PSD95 (cat#: ab18258, Abcam, USA), rabbit anti-synaptophysin (cat#: CY5273, Abcam, China), rabbit anti-drebrin (cat#: 10260-1-AP, Proteintech, USA). Subsequently, the sections were incubated with goat anti-rabbit IgG polymer labeled with biotin (cat#: SP-9001, ZSGB-BIO, China) for 30 min, horseradish enzyme-labeled streptomycin for 1 h, and DAB staining. The hippocampal CA1 region was observed and imaged under a 40× light microscope (Leica, Germany), and the average optical density was analyzed using Fiji software (National Institutes of Health, USA). Intra-assay coefficients of variation were 2.03% to 4.89%.
3
Immunohistochemical Staining Protocol
4
Immunohistochemical Analysis of Retinal Protein
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Retinas were freshly dissected and immediately placed in 10% formalin overnight. Relief cuts were made and the retinas were embedded in 5% agarose. Using a vibratome, 150 μm transverse sections were cut and the sections were floated in PBS. After blocking in 1% bovine serum albumin, 0.5% Triton X-100, and 2% normal donkey serum for 2–3 hours, sections were incubated in primary antibody overnight at 4° C. After washing in PBS, secondary antibodies were applied at room temperature for 1 hour. Sections were again washed and then mounted for confocal microscopy (LSM710, Carl Zeiss). Antibodies were as follows: 3R10 mouse anti-RS17 (link) (gift of Professor Robert Molday, 1:5); rabbit anti-GS (Sigma, 1:1000); rabbit anti-synaptophysin (abcam, 1:1000).
5
Immunohistochemical Analysis of Tau Proteins
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The fish were sacrificed, and the heads were fixed in 2% PFA overnight at 4 °C. After washing 3 times 10 min with Phosphate buffer, the heads were decalcified using 20% Sucrose/EDTA. Samples were mounted in 20% Sucrose/7.5% Gelatin as described39 (link). Samples were cryosectioned at 12 µM thickness, and were stained with the following antibodies: chicken anti-GFP IgG (1:2000; Abcam), mouse anti-Tau13 IgG1 (IHC 1:1000, WB 1:500; Abcam), mouse anti-phospho-Tau (AT180) IgG1 (IHC 1:500, WB 1:1000; Pierce, Thermo Scientific), mouse anti-phospho-Tau (AT8) IgG1 (IHC 1:500, WB 1:1000; Pierce, Thermo Scientific), mouse anti-phospho-Tau (AT270) IgG1 (IHC 1:500, WB 1:1000; Pierce, Thermo Scientific), mouse anti-PCNA IgG2a (1:500; Dako), rabbit anti-S100β IgG (1:500; Dako), rabbit-anti-L-plastin IgG (1:5000) (gift from Michael Redd), rabbit anti-beta actin (WB 1:1000, Abcam), rabbit anti-synaptophysin (1:500, Abcam), mouse anti-HuC/D IgG2b (1:500, Life Technologies), mouse anti IL4 (1:500, R&D Systems), and rabbit anti-Tau (T22) (1:500, Merck).
6
Protein Extraction from Brain Regions
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Cortex, hippocampi and cerebellum for protein extraction were minced in extraction buffer (50 mM Tris, 150 mM NaCl, 1% NP40 (Roche) and 1% Triton X-100 (Sigma) and incubated at 4 degrees C for 30 minutes. Synaptosome fractions were isolated using Syn-PER synaptic protein extraction reagent (ThermoFisher Scientific) according to the manufacturer’s instructions. Lysates and fractions were separated on Invitrogen Bolt™ precast 4–12% polyacrylamide gels and transferred to PVDF membrane before being blotted. Antibodies and their corresponding dilutions were: mouse anti-PCDH19 1:100 (Abcam, ab57510) and rabbit anti-β-ACTIN 1:1000 (Cell Signalling Technology, #4967), mouse anti-PSD95 1:2000 (ThermoScientific, 7E3-1138) and rabbit anti-SYNAPTOPHYSIN 1:5000 (Abcam, ab32127). Membranes were blocked in 5% BSA+5% Skim milk in Tris-buffered saline+0.1% Tween 20 (TBST) and antibodies were incubated with the membrane in 5% BSA in TBST. Membranes were developed using Bio-Rad Clarity Western ECL substrate and imaged using a Bio-Rad ChemiDoc.
7
Neuronal Cytoskeletal and Synaptic Protein Analysis
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Immunostaining was performed to examine morphological changes in the neuronal cytoskeletal and synaptic proteins of cortical neurons cultured for 3 days and 10 days, respectively. Neurons were fixed with 4% paraformaldehyde and 15% picric acid for 1 h at room temperature. Permeabilization of fixed cultures was performed by incubation for 1 h in incubation media containing 5% goat serum and 0.1% Triton X. Cells were then incubated with primary antibodies rabbit anti-synaptophysin (1:500, Abcam) and mouse anti-PSD95 (1:2,000; NeuroMab) to study the development of synaptic proteins. Primary antibodies were applied at 4°C overnight. Following 3× wash with 1× PBS, secondary antibodies AlexaFluor 488 goat anti-rabbit IgG (1:200; Invitrogen) and AlexaFluor 546 goat anti-mouse IgG (1:200; Invitrogen) were applied for 1 h at room temperature. Mouse anti-β-tubulin (1:500; Invitrogen) was used to study the development of cytoskeletal proteins. Preparations were washed 3× in 1× PBS and mounted with MOWIOL mounting media. A Zeiss confocal microscope (LSM 510 Meta, Zeiss, Germany) was used to take fluorescence images. Image acquisition parameters such as exposure times, gain settings, laser intensity, pinhole size, etc., remained the same between control and treated cultures.
8
Synaptic protein analysis by Western blot
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Proteins from isolated synaptosomes were loaded on 10% SDS-PAGE gel and then transferred to PVDF membranes, blocked in 5% nonfat milk, and incubated with primary antibodies, which included rabbit anti VGLUT1 (Synaptic Systems), mouse anti-PSD95 (Millipore), mouse anti-Gephyrin (Synaptic Systems), rabbit anti GluN2B (Synaptic Systems), rabbit anti-Synaptophysin (Abcam) overnight at 4°C. Lysates from microglia were incubated with the primary antibody rabbit anti PTEN (Cell Signaling Technology). Membranes were then incubated with HRP-conjugated secondary antibodies, and immunoblots were developed with enhanced chemiluminescence (Thermo Fisher Scientific). Images were analyzed with ImageJ software.
9
Western Blot Analysis of Synaptic Proteins
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According to a previously described method [13 (link),21 (link)], western blot was conducted. From the peri-infarct cortex and hippocampus, total protein was extracted using lysis buffer containing 150mM NaCl, 1mM phenylmethylsulfonyl fluoride, 1% Nonidet P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 100-mg/mL leupeptin, 50mM Tris-HCl (pH, 7.5). Protein was quantified using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). Protein, 30 µg, was separated on SDS polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Chicago, IL, USA). The membrane was blocked with skim milk, then membrane was incubated by mouse anti-β-actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit anti-PSD-95 antibody (1:1,000; Abcam, Cambridge, UK), rabbit antisynaptophysin (1:1,000; Abcam), rabbit antiBDNF antibody (1:1,000; Alomone Labs, Jerusalem, Israel), and rabbit anti-TrkB antibody (Santa Cruz Biotechnology) at 4°C during overnight. After washing, species appropriate horseradish peroxidase-conjugated secondary antibodies were incubated for 1 hour. Band detection was performed using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology).
10
Protein Expression Analysis of Brain Tissues in Ischemic Stroke Model
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Brain tissues were collected for rats enrolled into the 2 week treatment cohort at 35 days after MCAO (n = 5/Vehicle, n = 4/RPh201, and n = 4/Sham), and for rats enrolled into the 16 week treatment cohort at 133 days after MCAO (n = 5/Vehicle, n = 5/RPh201 treatment, and n = 4/Sham). Briefly, rats were decapitated under deep anesthesia. Brains were separated into left and right hemisphere and flash-frozen at −80°C for subsequent protein extraction. Total proteins were extracted for Western blots. The following primary antibodies were employed: rat anti-Myelin basic protein (MBP, Millipore), chicken anti-Neurofilament heavy chain (NFH, ThermoFisher Scientific), and rabbit anti-Synaptophysin (Abcam). The protein levels of MBP, NFH, and Synaptophysin were determined by Western blot analysis. Additional antibodies used for Western blot analysis were: Vascular endothelial growth factor receptor (VEGFR, Abcam), Intercellular adhesion molecule 1 (ICAM, Cell Signaling), Toll-like receptor-2 (TLR2, Abcam), and Toll-like receptor-4 (TLR4, Abcam).
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