IHC was conducted as previously described34 (link). IHC staining was evaluated by three senior pathologists using Image-Pro Plus software (NIH, USA). The antibodies used in the IHC staining were listed below: SPATS2 (sc-390306, Santa Cruz), ki-67 (27309-1-AP, Proteintech). Sections were semi-quantitatively scored for the SPATS2 IHC staining patterns as follows: the staining extent in each core was scored as 1 + (< 25% staining of cells), 2 + (25–50% staining of tumor cells), 3 + (50 to 75% staining of tumor cells), or 4 + (> 75% staining of tumor cells). Additionally, the staining intensity was quantified as 0 (negative), 1 + (weak), 2 + (intermediate), or 3 + (strong). The final score was obtained by multiplying the intensity and extension values (range 0–12) and the samples were grouped as 1 + (score 0-1), 2 + (score 2-3), 3 + (score 3-4), 4 + (score 6–8) and 5 + (score 9-12) staining. Meanwhile, for statistical purposes, scores of 4+ and 5+ were defined as high expression and the other final scores were considered as low expression.
Ki67 27309 1 ap
Manufactured by Proteintech
Sourced in United States, China
Ki67 (27309-1-AP) is a primary antibody produced by Proteintech for the detection of the Ki67 protein. Ki67 is a nuclear protein that is associated with cellular proliferation. It is a reliable marker of cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
α sma 55135 1 ap, by Proteintech (2 mentions)
Vimentin 10366 1 ap, by Proteintech (1 mentions)
Ab72100, by Abcam (1 mentions)
Pannoramic scan, by 3DHISTECH (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
Pkm2 15822 1 ap, by Proteintech (1 mentions)
Ldha 21799 1 ap, by Proteintech (1 mentions)
Glut1 21829 1 ap, by Proteintech (1 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Fibronectin 66042 1 ig, by Proteintech (1 mentions)
N cadherin 22018 1 ap, by Proteintech (1 mentions)
Smad2 3 sirna, by Santa Cruz Biotechnology (1 mentions)
β actin, by Proteintech (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Cleavage caspase 3, by Cell Signaling Technology (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Mouse anti rabbit igg hrp sc 2357, by Santa Cruz Biotechnology (1 mentions)
Anti gsdmd 20770 1 ap, by Proteintech (1 mentions)
Beta actin, by ABclonal (1 mentions)
17 protocols using ki67 27309 1 ap
1
Quantitative Immunohistochemical Analysis
2
IHC Staining of Colorectal Tissues
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IHC staining of colorectal tissues was performed on 5-µm-thick formalin-fixed and paraffin-embedded (FFPE) tissue sections according to the standard procedures as described previously [14 (link)]. The sections were stained using antibodies against CXCR7 (ab72100) (Abcam, USA), α-SMA (55135-1-AP), Vimentin (10366-1-AP), and Ki67 (27309-1-AP) (Proteintech, USA) according to the manufacturers’ instructions. The IHC results were taken with a digital slide scanning system (Pannoramic Scan, 3DHISTECH Ltd) and semiquantified of mean density (IOD/area) by image-pro plus 6 software (IPP, USA).
3
Immunohistochemical Analysis of NR5A2, GDF15, and Ki67
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Tissue microarray was purchased from Outdo Biobank (Shanghai, China) (HPan-Ade060CD-01). IHC analysis was performed with NR5A2 (#22460-1-AP, working dilution 1:5000, Proteintech), GDF15 (#27455-1-AP, working dilution 1:500, Proteintech), Ki67(27309-1-AP, working dilution 1:3000, Proteintech) antibodies. Two independent pathologists, who were uninformed about the patient data and histopathological features of the samples, were responsible for reviewing and scoring the degree of immunostaining separately. Staining intensity scoring was performed as described previously43 (link).
4
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemical staining was used to detect the expression of proliferation and metabolic indicators. Briefly, the subcutaneous tumor tissue was cut into 3um sections and then dewaxed. The sections were then incubated with the primary antibodies KI-67(27309-1-AP, Proteintech), PCNA (10205-2-AP, Proteintech), GLUT1 (21829-1-AP, Proteintech), PKM2(15822-1-AP, Proteintech), LDHA (21799-1-AP, Proteintech), HK2(66974-1-Ig, Proteintech) at 4°C overnight. After washing three times with PBS, each piece was stained with 3,3 ‘diaminobenzidine (DAB) and then observed and photographed under the light microscope (Nikon, Tokyo, Japan).
5
Immunohistochemical Profiling of Tissue Samples
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Paraffin-embedded tissue was cut into 5 μm thick slices, dewaxed with xylene, and hydrated with ethanol. Then, the slides were heated at 95°C in a 0.01 M citrate buffer (pH = 6.0) and quenched for peroxidase activity with 3% hydrogen peroxide for 20 min to retrieve antigens. After being treated with 10% goat serum, the slides were incubated overnight with antibodies at 4°C. PBS washing was followed by incubation with goat anti-rabbit IgG for 1 h and then stained with 3,3-diaminobenzidine. When all sections were dehydrated and sealed, we selected images with an inverted microscope (ZEISS). Immunohistochemistry was performed with the following antibodies: Ki67 (27309-1-AP; Proteintech), MALT1 (66225-1-Ig; Proteintech), p-p65 (AB11014, phosphor-Ser536), fibronectin (66042-1-Ig; Proteintech), N-cadherin (22018-1-AP; Proteintech), E-cadherin (20874-1-AP; Proteintech), vimentin (10366-1-AP; Proteintech) Snail1 (26183-1-AP; Proteintech), and Snail2 (12129-1-AP; Proteintech).
6
TGF-beta Signaling Pathway Regulation
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TGF-β was purchased from PeproTech, Inc. FXR1 (cat. no. 67813-1-Ig, 1:10,00 or 1:100), Ki67 (27309-1-AP; 1:100) and β-actin (cat. no. 66009-1-Ig, 1:10,00) antibodies were purchased from ProteinTech Group, Inc. The FXR1 antibody was diluted 1:1,000 in the western blotting assay and 1:100 in immunohistochemistry assays. SMAD2/3 (cat. no. # 8685S, 1:1,000), N-Cadherin (cat. no. # 4061, 1:1,000), and slug (cat. no. 9585S, 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc. The secondary antibodies used were HRP-conjugated Affinipure Goat Anti-Rabbit (1:2,000 or 1:50; cat. no. SA00001-2) and HRP-conjugated Affinipure Goat Anti-Mouse (1:2,000; cat. no. SA00001-1) (both from ProteinTech Group, Inc.). HRP-conjugated Affinipure Goat Anti-Rabbit was diluted 1:2,000 in the western blotting assay and 1:50 in immunohistochemistry assays. SMAD2/3 siRNA was purchased from Santa Cruz Biotechnology, Inc. (cat. no. sc-37238). shRNA was purchased from Public Protein/Plasmid Library. The sequences of the FXR1 knockdown shRNAs were: shFXR1#1, 5′-GCTAGAGGTTTCTTGGAATTT-3′; shFXR1#2, 5′-CGCCAGGTTCCATTTAATGAA-3′; and negative control (shNC), 5′-GTTCTCCGAACGTGTCACGTT-3′. The FXR1 expression plasmid with a 3′FLAG tag was purchased from Public Protein/Plasmid Library.
7
Evaluating ALKAL1 Expression in Colorectal Cancer
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Immunohistochemical staining was performed on 4 μm tissue sections using an EnVision™ Kit (DAKO, Denmark). After deparaffinized, rehydrated in graded ethanol, antigen retrieval and blocking, the slides were incubated overnight at 4 °C in a humidified chamber with anti-ALKAL1 antibodies (Thermo Fisher, PA5-55591), Vimentin (sc-373717), Ki67 (27309-1-AP, Proteintech) and caspase 3 cleavage (Asp175, 9661, Cell Signaling Technology) diluted 1:50 in PBS. Scores given by two independent Pathologists were averaged for further comparative evaluation of ALKAL1 expression. The details of scoring criteria were described in our previous study 26 (link), 27 . According to this method, ALKAL1 expression in colorectal cancer samples was evaluated by the staining index, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. High or low expression of ALKAL1was stratified according to the following criteria: SI ≤ 4 was defined as tumors with low expression of ALKAL1, and SI ≥ 6 was defined as tumors with high expression of ALKAL1.
8
Western Blot Antibody Inventory
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Antibodies specific for DYKDDDDK (FLAG tag) (#14,793), LCN2 (#44,058), p-EGFR (Tyr1173, #4407), ERK (#4696), p-ERK (#4370) and mouse IgG-HRP (#7076) were purchased from Cell Signaling Technology. Antibodies specific for GAPDH (sc-47724) and β-tubulin (sc-166729) and mouse anti-rabbit IgG-HRP (sc-2357) were purchased from Santa Cruz Biotechnology. Antibodies specific for ERK1/2 (YT1625) and p-ERK1/2 (YP0101) were purchased from ImmunoWay. Antibodies specific for LCN2 (26,991–1-AP), EGFR (66,455–1-Ig, 51,071–2-AP), and KI67 (27,309–1-AP) were purchased from Proteintech.
9
Immunohistochemical Analysis of DNA Damage
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Briefly, tissues were fixed with 10% formalin and embedded in paraffin, 4 μm paraffin embedded were performed and stained with H&E or IHC. The primary antibodies used were Ki67 (27309–1-AP, Proteintech, 1:200), γH2AX (1:100) and RAD51 (1:500). Major organ sections and primary tumors were performed with H&E staining. Images were collected using Microscope (BX53F2, Olympus, Tokyo, Japan) and IHC stained images were analyzed by Image J 1.8.0.
10
Antibody Validation and Normalization
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Anti-CD147 (66290-1-lg), Ki-67(27309-1-AP), and anti-GSDMD (20770-1-AP) antibodies were supplied by Proteintech Group (Proteintech, USA). A-tubulin and beta-actin were purchased from Abclonal (Abclonal, China). All reagents were purchased from Sigma (St Louis, MO, USA).
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