2 weeks post‐surgery, four rats from each group were sacrificed by intraperitoneal pentobarbital injection (25%, lethal dose). Subsequently, the nerve grafts were harvested and cut longitudinally into 7 µm‐thick sections using a freezing microtome (CM3050S; Leica Microsystems). Axons were then visualized using an anti‐neurofilament NF200 antibody (1:200; N0142; Sigma‐Aldrich) and a secondary Alexa Fluor 488‐conjugated goat anti‐rat IgG antibody (1:1000; A11006; Invitrogen). Additionally, Schwann cells were visualized using an S100b antibody (1:200; HPA015768; Sigma‐Aldrich) and a secondary antibody Alexa Fluor 594‐conjugated donkey anti‐rabbit IgG (1:1000; R37119; Invitrogen). Sema5b expression was visualized using the S100b antibody (1:200; PA5‐66850; Thermo Fisher Scientific), and the secondary antibody Alexa Fluor 488‐conjugated donkey anti‐rabbit IgG (1:1000; R37118; Invitrogen). These dually stained sections were observed under a fluorescence microscope (Leica Q500MC; Leica, Germany).
Hpa015768
Manufactured by Merck Group
Sourced in United Kingdom
HPA015768 is a laboratory equipment product manufactured by Merck Group. It is designed to perform a specific function within a laboratory setting. The core function of this product is to facilitate precise and efficient laboratory processes, but a detailed description of its intended use is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
A11006, by Thermo Fisher Scientific (1 mentions)
Cm3050, by Leica (1 mentions)
Q500mc, by Leica (1 mentions)
R37119, by Thermo Fisher Scientific (1 mentions)
N0142, by Merck Group (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
Matrigel, by Corning (1 mentions)
G3893, by Merck Group (1 mentions)
M7020, by Agilent Technologies (1 mentions)
Ab19857, by Abcam (1 mentions)
Hpa014784, by Merck Group (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Target retrieval solution ph 9, by Agilent Technologies (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Ab731, by Abcam (1 mentions)
Biozero 8100 confocal microscope, by Keyence (1 mentions)
4 protocols using hpa015768
1
Histological Analysis of Nerve Graft Axons
2
Comprehensive Immunofluorescence Characterization of iPSCs, NPCs, and Astrocytes
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iPSC, NPC and astrocyte cultures were first plated in Matrigel (Corning, USA)‐coated Lab‐tek 8‐well chamber slide systems (Thermo Fisher, USA) at a density of 4 × 104 cells per well, then fixed with 4% PFA, followed by permeabilization with 0.01% Triton X‐100, and blocking with 5% BSA in PBS. The cells were then incubated overnight with the following primary or conjugated antibodies: rabbit anti‐OCT4 (ab19857, Abcam, 1:100); APC anti‐SSEA4 (FAB1435A, R&D Systems, 1:100); AF647 anti‐nestin (560,393, BD, 1:100); mouse anti‐GFAP (G3893, Sigma, 1:100); mouse anti‐Vimentin (M7020, DAKO, 1:100); rabbit anti‐S100‐β (HPA015768, Sigma, 1:100); rabbit anti‐AQP4 (HPA014784, Sigma, 1:100) at 4°C, and subsequently incubated with the following secondary antibodies: AF488 goat anti‐rabbit IgG (A11034, Life, 1:250); AF488 goat anti‐mouse IgG (A11001, Life, 1:250); AF594 donkey anti‐mouse IgG (A21203, Life, 1:250); AF594 donkey anti‐rabbit IgG (A21207 Life, 1:250) for 1h at room temperature. Slides were mounted with Vectashield containing DAPI (Vector Laboratories, USA), dried, sealed, and the analysis was performed using a Zeiss LSM 800 confocal microscope. Quantification of the percentage of positive cells was performed using ImageJ software.
3
Immunofluorescent Staining of Skin Tissue
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Skin tissue samples were fixed in 10% formaldehyde and embedded in paraffin. These were sliced into 5 μm sections, deparaffinized, dehydrated, and heat-incubated with antigen retrieval solution (Target Retrieval Solution, PH9; Dako, Agilent, Santa Clara, CA, USA) for approximately 15 min. After blocking with horse serum (Vector Laboratories, Marion, Burlingame, CA, USA), sections were incubated with primary antibodies specific for CD1a (1:100 dilution, #M357101, Dako, Santa Clara, CA, USA), Melan-A (1:100 Dilution, #ab731, Abcam, Cambridge, UK), and S100B (1:100 Dilution, #HPA015768, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C and then incubated with a secondary antibody for 1 h (1:500 dilution; anti-mouse IgG1 AlexFluor 488 for CD1a, anti-mouse IgG2b AlexFluor 555 or anti-mouse IgG2b AlexFluor 647 (pseudocolor white) for Melan-A, anti-rabbit IgG AlexFluor 555 for S100B; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) [22 (link)]. Sections were counterstained with Hoechst 33342 at a 1:500 dilution (Invitrogen, Carlsbad, CA, USA), and stained sections were visualized using a Biozero 8100 confocal microscope (Keyence Corporation, Osaka, Japan).
4
Brain Section Immunostaining Protocol
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All experiments were carried out at room temperature unless otherwise specified. After two-photon imaging, the mice were killed by intraperitoneal injection of Sleep-Away and perfused transcardially as described in the retrograde tracing section. Coronal and sagittal sections (40 μm) of whole brain were cut by using a sliding microtome frozen by powdered dry ice. Free-floating serial sections (taken one every third) were washed in TBS, incubated for 30 min in a blocking solution containing 4% normal horse serum (vol/vol), 1% BSA in TBS with 0.2% Triton X-100 (TBS-Tx) and incubated overnight at 4 °C with rabbit anti S100b primary antibody (HPA015768, Sigma) diluted 1:500 in the blocking solution mentioned above. Sections were then washed in TBS and incubated for 2 h with Alexa Fluor-labelled donkey anti-rabbit IgG secondary antibody (Alexa Fluor -555) diluted 1:500 in TBS-Tx. After washing three times for 10 min, sections were mounted onto chrom-alum/gelatin-coated slides, and air-dried for 2 h in the dark. Slides were cover-slipped by water-soluble glycerol-based mounting medium containing DAPI, and sealed with nail polish. As described above, Kruskal–Wallis ANOVA and post hoc contrasts (Dunn's Test) were used to test for an effect of distance from the prism face on cell density (DAPI+) and gliosis (S100b+). The results are reported in Supplementary Fig. 1c,d .
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