G6171

Manufactured by Merck Group

Sourced in United States

The G6171 is a laboratory equipment product from Merck Group. It is designed to perform a specific core function, but a detailed and unbiased description is not available at this time.

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Lab products found in correlation

7 protocols using g6171

Immunohistochemical Analysis of Nervous System Markers

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For the immunohistochemical analysis, paraffin-embedded sections were dewaxed, hydrated, and blocked with 5% bovine serum albumin (SW3015, Solarbio) for 30 min. Then, the sections were incubated with the following primary antibodies at 4 °C overnight: rabbit polyclonal anti-rat/human myelin basic protein (MBP) (1:200, 78896; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-rat/human S100 (1:200, ab868; Abcam, Cambridge, UK), mouse monoclonal anti-rat/human GFAP (1:400, G6171; Sigma-Aldrich, St. Louis, MO, USA), rabbit monoclonal anti-human MBP (1:400, ab133620; Abcam, Cambridge, UK), rabbit polyclonal anti-human S100 (1:300, ab15520; Abcam, Cambridge, UK), and mouse monoclonal anti-human GFAP (1:400, ab8975; Abcam, Cambridge, UK). Subsequently, the sections were rinsed with PBS and incubated with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit secondary antibodies for another 2 h at 37 °C. Finally, the sections were stained with a DAB (3,3′-diaminoben zidine) Kit (Zsbio, Beijing, China) and observed by fluorescence microscope (Axiovert A1, Carl Zeiss, Germany). Areas of positive staining were determined using the Image J software.

Luo L., He Y., Jin L., Zhang Y., Guastaldi F.P., Albashari A.A., Hu F., Wang X., Wang L., Xiao J., Li L., Wang J., Higuchi A, & Ye Q. (2020). Application of bioactive hydrogels combined with dental pulp stem cells for the repair of large gap peripheral nerve injuries. Bioactive Materials, 6(3), 638-654.

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Comprehensive Immunoblotting of Mitochondrial and Cellular Markers

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Immunoblotting was performed with anti-CPT1A (1/1,000) (ab128568; Abcam), anti-CPT1B (1/1,000) (ab134988; Abcam), anti-CPT1C (1/1,000) (ab87498; Abcam), anti-CTP2 (1/1,000) (ab181114; Abcam), anti-heat-shock protein-60 (HSP60) (1/1,000) (ab46798; Abcam), anti-TOMM20 (1/1,000) (ab56783; Abcam), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-UQCRC2 (1/1,000) (ab14745; Abcam), anti-GFAP (1/500) (G6171; Sigma), anti-NDUFB8 (1/1,000) (ab110242; Abcam), anti-NDUFA9 (1/1,000) (ab14713; Abcam), anti-SDHA (1/1,000) (ab14715; Abcam), anti-MTCO1 (1/1,000) (ab14705; Abcam), anti-COX IV (1/1,000) (ab16056; Abcam), anti-Iba1 (1/1,000) (019-19741; Wako), anti-MAP2 (1/1,000) (ab32454; Abcam), anti-OLIG2 (1/1,000) (ab109186; Abcam), anti-PDHA1 (1/1,000) (no. 3205; Cell Signaling), anti-phosphoSer²⁹³-PDHA1 (1/1,000) (no. 31866; Cell Signalling), anti-β-Tubulin III (1/300) (T2200; Sigma) and anti-β-actin (1/30,000) (A5441; Sigma).

Morant-Ferrando B., Jimenez-Blasco D., Alonso-Batan P., Agulla J., Lapresa R., Garcia-Rodriguez D., Yunta-Sanchez S., Lopez-Fabuel I., Fernandez E., Carmeliet P., Almeida A., Garcia-Macia M, & Bolaños J.P. (2023). Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition. Nature Metabolism, 5(8), 1290-1302.

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Immunoblotting Analysis of Mitochondrial Proteins

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Immunoblotting was performed with anti- C-subunit of ATP synthase (SCMAs) (1/1000) (ab181243; Abcam), anti-VDAC (1/666) (PC548; Calbiochem), anti-heat-shock protein-60 (HSP60) (1/666) (ab46798; Abcam), anti-PINK1 (1/500) (sc-33796; Santa Cruz Biotechnology), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-CDK5 (1/500) (sc-6247; Santa Cruz Biotechnology), anti-PFKFB3 (1/500) (H00005209-M08; Novus Biologicals), anti-p25/35 (1/666) (2680; Cell Signalling), anti-caspase-3 (1/2000) (9661S; Cell Signalling), anti-CLN7 (1/500) (donated by Dr. Stephan Storch), anti-Parkin (1/100) (sc-32282; Santa Cruz Biotechnology), anti-LC3B (1/1000) (2775; Cell Signaling), anti-GFAP (1/500) (G6171; Sigma), anti-β-Tubulin III (1/500) (ab18207; Abcam) and anti-β-Actin (1/30,000) (A5441; Sigma).

Lopez-Fabuel I., Garcia-Macia M., Buondelmonte C., Burmistrova O., Bonora N., Alonso-Batan P., Morant-Ferrando B., Vicente-Gutierrez C., Jimenez-Blasco D., Quintana-Cabrera R., Fernandez E., Llop J., Ramos-Cabrer P., Sharaireh A., Guevara-Ferrer M., Fitzpatrick L., Thompton C.D., McKay T.R., Storch S., Medina D.L., Mole S.E., Fedichev P.O., Almeida A, & Bolaños J.P. (2022). Aberrant upregulation of the glycolytic enzyme PFKFB3 in CLN7 neuronal ceroid lipofuscinosis. Nature Communications, 13, 536.

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Immunofluorescence Staining of Neural Markers

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Similar to IHC described above, immunofluorescent detection was implemented to the serial sections with appropriate combinations of the following primary antibodies and dilutions: rabbit GFP (ab3080, Millipore, 1:200), mouse GFP (a‐11120, Thermo, 1:100), rabbit GFP (ab290, Abcam, 1:1000), rabbit Caspase3 (AF835, R&D Systems, 1:100), rat BrdU (ab6326, Abcam, 1:800), mouse BrdU (Bu20a, Cell Signaling, 1:500), mouse Nestin (MAB353, Millipore, 1:50), rabbit PDGFRa (AB61219, Abcam, 1:200), rabbit NG2 (ab5320, Millipore, 1:50), mouse MBP (NE1018, Millipore, 1:100), mouse Olig2 (MABN50, Millipore, 1:200), rabbit Nogo‐A (AB5888, Millipore, 1:200), mouse BCAS1 (sc‐393808, Santa Cruz, 1:200), rabbit GFAP (G6171, Sigma, 1:100), rabbit DCX (ab18723, Abcam, 1:100), mouse NeuN (MAB377, Millipore, 1:200), rabbit Connexin29 (34–4200, Thermo, 1:50), mouse Connexin32 (MAB3069, Chemicon, 1:100), and rabbit Connexin47 (36–4700, Thermo, 1:100). Accordingly, the secondary antibodies and dilutions applied were the following: goat anti‐rabbit Alexa Fluor 488 (a11008, 1:500), goat anti‐mouse Alexa Fluor 488 (a11001, 1:500), goat anti‐rat Alexa Fluor 555 (a21434, 1:500), goat anti‐rabbit Alexa Fluor 647 (ab150079, 1:500), and goat anti‐mouse Alexa Fluor 647 (a21235, 1:500). Sections were then mounted with 4’,6‐Diamidino‐2‐Phenylindole (DAPI; D1306, Invitrogen) and slides were coverslipped.

Theotokis P., Kesidou E., Mitsiadou D., Petratos S., Damianidou O., Boziki M., Chatzidimitriou A, & Grigoriadis N. (2021). Lumbar spine intrathecal transplantation of neural precursor cells promotes oligodendrocyte proliferation in hot spots of chronic demyelination. Brain Pathology, 32(4), e13040.

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Spinal Cord Protein Analysis Protocol

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After the 13-Day experiment, all mice were anesthetized with 2% isoflurane, and the right side of lumbar 4–6 spinal cord was harvested. Total protein was extracted with Radio-Immunoprecipitation Assay (RIPA) buffer. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5%, 10%, or 15% gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in blocking buffer (5% skim milk) for 2 h before being incubated overnight at 4°C with primary antibodies, including TRPV1 (1:3000, 66983-1-Ig, Proteintech Group, Inc.), NMDAR2B (1:1000, D8E10, Cell Signaling Technology, Inc.), IBA-1 (1:1000, AB5076, Abcam, Inc.), GFAP (1:500, G6171, Sigma, Inc.), β-actin (1:1500, D6A8, Cell Signaling Technology, Inc.), and GAPDH (1:1500, D16H11, Cell Signaling Technology, Inc.). The membranes were washed three times with Tris-Buffered Saline and Tween-20 (TBST) for 10 min each and then incubated with secondary antibodies, such as horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (1:5000, Cat. No. SA00001-2, Proteintech Group, Inc.) and HRP-conjugated AffiniPure goat anti-mouse IgG (1:5000, Cat. No. SA00001-1, Proteintech Group, Inc.). Finally, the bands were visualized using Enhanced Chemiluminescence (ECL) substrates, and the intensity analysis was conducted using ImageJ software.

Fang K., Lu P., Cheng W, & Yu B. (2024). Kilohertz high-frequency electrical stimulation ameliorate hyperalgesia by modulating transient receptor potential vanilloid-1 and N-methyl-D-aspartate receptor-2B signaling pathways in chronic constriction injury of sciatic nerve mice. Molecular Pain, 20, 17448069231225810.

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GFAP Immunofluorescence Staining Protocol

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Cells cultured on coated glass coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and washed with PBS as described previously6 (link). A mouse monoclonal antibody against GFAP (Sigma, #G-6171) was used as a primary antibody. Alexa 555- or Alexa 647-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were used for visualization. For simultaneous labeling experiments, immunostaining was performed after FISH.

Ito K., Sanosaka T., Igarashi K., Ideta-Otsuka M., Aizawa A., Uosaki Y., Noguchi A., Arakawa H., Nakashima K, & Takizawa T. (2016). Identification of genes associated with the astrocyte-specific gene Gfap during astrocyte differentiation. Scientific Reports, 6, 23903.

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Huntingtin Protein Immunohistochemistry in Mouse Brain

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Animals were transcardially perfused with 4% paraformaldehyde in Phosphate-Buffered Saline (PBS) solution (pH 7.4). The brain was rapidly dissected out, post-fixed o/n at 4°C in the same fixative and then kept at 4°C in 30% sucrose/PBS until it had sunk. Using a vibratome (Vibratome 1000 Plus Sectioning System, St Louis, MO, USA), coronal sections of 40 μm thickness were cut throughout the striatum (−3 mm to +3 mm from Bregma) and every 10th sections samples were processed for immunohistochemistry. To block endogenous peroxidase free-floating sections were treated for 10 min with Tris-Buffered Saline (TBS) solution containing 10% methanol and 3% H₂O₂. After washing in TBS, sections were incubated for 1 h in blocking solution (1% BSA, 10% FCS, 1% fish gelatin) and then o/n at 4°C with the primary antibodies in TBS containing 1% BSA, 0.3% Triton X-100 and 0.1% fish gelatin. Sections were washed and incubated for 1 h with secondary anti-mouse Alexa-488 conjugated antibody (1:2000). Images were captured using a Leica TCS SP2 confocal microscope.
Primary antibodies were: EM48 anti-huntingtin (1:100, MAB5374, Chemicon), anti-glial fibrillary acidic protein (GFAP; 1:1000, G6171, Sigma) and anti-γH2AX (1:500, 05–636, Millipore).

Agostoni E., Michelazzi S., Maurutto M., Carnemolla A., Ciani Y., Vatta P., Roncaglia P., Zucchelli S., Leanza G., Mantovani F., Gustincich S., Santoro C., Piazza S., Del Sal G, & Persichetti F. (2016). Effects of Pin1 Loss in HdhQ111 Knock-in Mice. Frontiers in Cellular Neuroscience, 10, 110.

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