Paraffin-embedded sections from primary tumor of 102 patients with lymph node metastasis were deparaffinized in xylene and hydrated in decreasing concentrations of ethyl alcohol. The sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity, and heated for 10 min at 105 °C by autoclave in Target Retrieval Solution (DAKO, Carpinteria, CA, USA). Nonspecific binding was blocked by incubating with 10% normal rabbit serum for 10 min. The specimens were incubated with anti-GLUT1 antibodies (sc-377228; 1:150; Santa Cruz, Dallas, TX, USA; RRID: AB_2716767) for 30 min at room temperature, anti-PKM2 antibodies (sc-365684; 1:200; Santa Cruz; RRID: AB_10844484) at 4 °C overnight, anti-PDH-E1α antibodies (sc-377092; 1:100; Santa Cruz; RRID: AB_2716767)35 (link) at 4 °C overnight, and with CA9 antibodies (NB100–417; 1:1000; Novus Biologicals, Centennial, CO, USA; RRID: AB_10003398) for 30 min at room temperature. These sections were incubated with a mouse linker for 10 min and peroxidase-labeled polymer (Histofine SAB-PO(M) kit, Nichirei Biosciences Inc., Tokyo, Japan) for 5 min, followed by counterstaining with Mayer’s hematoxylin.
Sc 365684
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-365684 is a laboratory instrument designed for cell culture and biochemical applications. It provides controlled temperature and humidity conditions to maintain optimal cell growth and experimental environments. The core function of this product is to facilitate controlled incubation of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Target retrieval solution, by Agilent Technologies (2 mentions)
Ab23750, by Abcam (2 mentions)
Ab14683, by Abcam (2 mentions)
Ab5694, by Abcam (2 mentions)
Nb100 417, by Novus Biologicals (1 mentions)
Sc 377092, by Santa Cruz Biotechnology (1 mentions)
Sc 377228, by Santa Cruz Biotechnology (1 mentions)
Histofine sab po m kit, by Nichirei Biosciences (1 mentions)
Ab19304, by Abcam (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Ab175933, by Abcam (1 mentions)
Polyperoxidase anti rabbit igg, by ZSGB-BIO (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab104836, by Abcam (1 mentions)
Ab53066, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab32148, by Abcam (1 mentions)
Ab74109, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab13492, by Abcam (1 mentions)
6 protocols using sc 365684
1
Immunohistochemical Analysis of Tumor Metabolism Markers
2
Immunohistochemical and Immunofluorescence Analysis of Tumor Markers
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Tumor tissues were fixed with 4% paraformaldehyde and then sectioned into 4 μm of sections. IHC was performed according to protocols of the manufacturor. The sections were incubated with rabbit anti-MMP2, MMP9, p-ATM, GLUT1, PKM2 and TGFβ1 polyclonal antibody (1:200, Bioworld) overnight at 4 °C. Then, the sections were sequentially incubated with polyperoxidase-anti-rabbit IgG (ZSBiO) for 30 min at 37 °C, then stained with diaminobenizidine.
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
3
Western Blotting Analysis of Cellular Proteins
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Western blotting analysis was performed as described previously [11 (link)]. Briefly, total cell proteins were obtained using RIPA lysis buffer (P0013B, Beyotime, China), quantified with the BCA protein assay kit (P0012, Beyotime). 50 μg of total proteins were separately electrophoresed in 8%–12% SDS-PAGE gel, subsequently incubated with appropriate primary antibodies as followings: FN (ab23750, abcam,1:1000), FAP (ab53066, abcam,1:1000), α-SMA (ab5694, abcam,1:1000), ATM (2873, CST, 1:1000), p-ATM (5883, CST, 1:1000), γH2AX (9718, CST, 1:1000), CHK2-T68 (ab32148, abcam, 1:1000), Na+/K+ ATPase (ab58457, abcam, 1:800), Hsp90 (ab13492, abcam, 1:800), AKT (4685, CST, 1:1000), p-AKT (12694 s, CST, 1:1000), GLUT1 (ab14683, abcam, 1:500), p-ST/Q (6966 s, CST, 1:1000), PKM2 (sc365684, Santa Cruz, 1:500), MCT4 (ab74109,1:1000), MCT1 (ab90582,1:1000) TGFβ1 (ab675195, abcam, 1:1000), P38 (bs4635, bioworld, 1:1000), p-P38 (bs3566, bioworld, 1:1000), MMP2 (ab92538, abcam, 1:800), and MMP9 (ab76003, abcam, 1:800), GLUT3 (ab41525,1:800), HK2 (ab104836,1:800), HPI (ab86950,1:1000), LDHA (ab101562,1:1000). The appropriate horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG (ZSGBBIO, China) was used as secondary antibodies. The protein bands were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan).
4
Interactome Capture of SHP1, PKM2, LDHB
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The SHP1, PKM2 and LDHB interactome was captured from Jurkat cells using rabbit SHP1 (1:50; 24546-1-AP, Proteintech), and mouse PKM2 (1:50; sc-365,684, Santa Cruz Biotechnology) and LDHB antibodies (1:50; sc-100,775, Santa Cruz Biotechnology). Mouse IgG antibodies (abs955, Absin) were used as a control for non-specific binding proteins. Protein A/G agarose beads (10 µl) was washed twice with 200 µl PBS buffer, and incubated with 1 µl antibody prepared in PBS at room temperature for 30 min on a mixer. The lysed cells were centrifuged for 2 min at 12,000 × g and the supernatant was collected. The entire supernatant was mixed with 10 µl of beads with antibody and incubated at 4 °C on a rocker platform overnight. The beads were centrifuged for 1 min at 12,000 × g and the supernatant was discarded. The beads were washed three times with 1 ml of cold wash buffer. The beads were then resuspended in 60 µl of electrophoresis sample buffer and heated to 95 °C for 5 min. The beads were centrifuged for 1 min at 12,000 × g and the supernatant collected and used for MS analysis or western blotting.
5
Immunohistochemical Analysis of Endometrial Proteins
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Immunohistochemical staining was performed as described previously (43 (link)). Human endometrium and decidua were labeled with rabbit anti-human FBP1, PFK1, IL-27, and PKM antibodies (Abs) (dilution 1:100; sc-271241, sc-377346, sc-390482, and sc-365684, Santa Cruz Biotechnology); uterine tissues of pregnant mice were labeled with rabbit anti-mouse CK7 Abs (10 μg/ml; ab9021, Abcam) by immunohistochemical staining.
6
Immunohistochemical Analysis of GLUT1 and PKM2 in NSCLC
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Immunohistochemical staining was performed on paraffin-embedded sections of primary lesions obtained from 445 patients with NSCLC. We deparaffinize the slides with a thickness of 4 µm in xylene and hydrated in decreasing concentrations of ethyl alcohol and incubate the sections with 3% hydrogen peroxide to block endogenous peroxidase activity. Then, we heated the sections in Target Retrieval Solution (DAKO, Carpinteria, CA, USA) for 10 min at 105°C using an autoclave. Nonspecific binding was blocked by incubating the sections with 10% normal rabbit serum for 10 min. The specimens were incubated with anti-GLUT1 antibodies (sc-377228; 1:150; Santa Cruz Biotechnology, Dallas, TX, USA; RRID: AB_2716767) for 30 min at 24°C and with anti-PKM2 antibodies (sc-365684; 1:200; Santa Cruz Biotechnology; RRID: AB_2716767) at 4°C overnight. Subsequently, these sections were incubated with a mouse linker for 10 min, and a peroxidase-labeled polymer solution (Histofine SAB-PO(M), #424022, Nichirei Biosciences Inc., Tokyo, Japan) for 5 min, then counterstained with Mayer's hematoxylin (#131-09665; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 30 sec at 24°C.
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