Standard hematoxylin and eosin (H&E) staining and dual-color immunofluorescence analysis using specific primary antibodies for mice F4/80 (1:100, MCA497GA, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and arginase (1:1000, D4E3M, Cell Signaling Technology, Danvers, MA, USA) were performed as per previously described methods [29 (link)].
D4e3m
Manufactured by Cell Signaling Technology
Sourced in United States
The D4E3M is a primary antibody that recognizes the phosphorylated form of the ERK1/2 protein. It can be used to detect the activated, phosphorylated state of ERK1/2 in various applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
4 protocols using d4e3m
1
Immunohistochemical Analysis of Macrophages
2
Immunohistochemical Analysis of Macrophages
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Standard hematoxylin and eosin (H&E) staining and dual-color immunofluorescence analysis using specific primary antibodies for mice F4/80 (1:100, MCA497GA, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and arginase (1:1000, D4E3M, Cell Signaling Technology, Danvers, MA, USA) were performed as per previously described methods [29 (link)].
3
Immunolabeling Protocol for Cytoskeletal and M1/M2 Markers
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Cells grown on 12 mm glass coverslips in a 24-well plate at a density of 15 × 103 cells/well, were fixed after treatments with 4% paraformaldehyde for 30 min, followed by treatment with 0.1 M glycine in PBS for 20 min. A total of 0.1% Triton X-100 in PBS was added for 5 min to allow permeabilization. To analyze cytoskeletal actin organization, cells were labeled with rhodamine-conjugated phalloidin (TRITC-phalloidin—Sigma-Aldrich, St. Louis, MO, USA) for 45 min. For the detection of M1/M2 polarization markers, cells were incubated with primary antibodies raised against rabbit polyclonal IgG anti-Iba1 (dil. 1:100—AB-83747, Immunological Sciences, Roma, Italy), rabbit polyclonal IgG anti-iNOS (dil. 1:100—D6B65, Cell Signaling Technology, Danvers, MA, USA), or rabbit polyclonal IgG anti-Arg-1 (dil. 1:50—D4E3M, Cell Signaling Technology, Danvers, Ma, USA), and subsequently with anti-rabbit Alexa Fluor 488 secondary antibodies. Finally, the cells were marked with DAPI to highlight the nucleus. The fluorescence signal was analyzed using an Axio Observer inverted microscope, equipped with the ApoTome System (Carl Zeiss Inc., Ober Kochen, Germany). Cell area was quantified with ImageJ software.
4
Immunohistochemical Profiling of HCC
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Pathologic slides from paraffin blocks prepared using surgical tissue specimens were stained with monoclonal antibodies against the following: Arg1 (1:500; catalog No. 93668; D4E3M; monoclonal rabbit anti-human; Cell Signaling Technology, Inc., USA); GS (1:1000; catalog No. ab176562; EPR13022(B); monoclonal rabbit anti-human; Abcam plc, USA); cytokeratin 19 (CK19) (1:500; catalog No. ab52625; EP1580Y; monoclonal rabbit anti-human; Abcam plc); EpCAM (1:500; catalog No. EM1111; 3-E1-E2; monoclonal mouse anti-human; HUABIO, China); and TP53 (catalog no. MAB-0674; MX008; monoclonal mouse anti-human; Maxim, China).
The immunostaining results were interpreted by three experienced pathologists using a blinded method. GS staining was recorded as positive (GS+) if it was equal to or greater than that of the hepatocytes surrounding the central vein in corresponding adjacent normal tissues.[18 (link)] Similarly, tumor specimens were recorded as Arg1+ if the staining was equal to or greater than that of adjacent normal tissues. We identified expression of GS and Arg1 in each HCC patient and divided all patients into four subgroups: GS−Arg1−, GS−Arg1+, GS+Arg1−, and GS+Arg1+. CK19 and EpCAM were regarded as positive if >5% of tumor cells showed membrane staining.[22 (link),23 (link)] Cases with moderate or strong nuclear staining in >5% of the cells were considered TP53 positive.
The immunostaining results were interpreted by three experienced pathologists using a blinded method. GS staining was recorded as positive (GS+) if it was equal to or greater than that of the hepatocytes surrounding the central vein in corresponding adjacent normal tissues.[18 (link)] Similarly, tumor specimens were recorded as Arg1+ if the staining was equal to or greater than that of adjacent normal tissues. We identified expression of GS and Arg1 in each HCC patient and divided all patients into four subgroups: GS−Arg1−, GS−Arg1+, GS+Arg1−, and GS+Arg1+. CK19 and EpCAM were regarded as positive if >5% of tumor cells showed membrane staining.[22 (link),23 (link)] Cases with moderate or strong nuclear staining in >5% of the cells were considered TP53 positive.
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