Carrying on in vivo suppression assays, the other half of spleen tissue was fixed immediately in 4% paraformaldehyde, and tissue sections were deparaffinized with xylene and rehydrated through graded ethanol washes. Heat mediated antigen retrieval was conducted by boiling in Tris/EDTA buffer (pH 9.0) for 5 min. The slides were incubated with 3% hydrogen peroxide for 20 min to eliminate endogenous peroxidase and then with 10% normal goat serum at room temperature in order to block any non-specific binding. After removing excess blocking buffer, the following primary antibodies, concerning rabbit anti-human CD8 antibody (ab93278, Abcam, Cambridge, UK) and mouse anti-human CD4 antibody (ZM-0418, ZSGB-BIO, Beijing, China), were diluted to the manufacturer’s recommendations and incubated in a humidified chamber at 4 °C overnight. The HRP-conjugated goat anti-rabbit IgG antibody (PV-6001, ZSGB-BIO, Beijing, China) and goat anti-mouse IgG antibody (PV-6002, ZSGB-BIO, Beijing, China) were used as the secondary antibody at 37 °C for 30 min. Finally, staining of the tissue sections was performed with an enhanced HRP-DAB chromogenic substrate kit (TIANGEN Biotech CO., LTD., Beijing, China). The sections were then counterstained with hematoxylin and visualized under a light microscope (Nikon, Tokyo Japan).
Zm 0418
Manufactured by ZSGB-BIO
Sourced in China
The ZM-0418 is a laboratory instrument designed for high-performance liquid chromatography (HPLC) analysis. It provides precise and efficient separation, identification, and quantification of various chemical compounds within a sample. The core function of the ZM-0418 is to facilitate the analysis of complex mixtures, enabling researchers and analysts to gain valuable insights into the composition and properties of their samples.
Automatically generated - may contain errors
Lab products found in correlation
Za 0508, by ZSGB-BIO (3 mentions)
Ab93278, by Abcam (2 mentions)
Enhanced hrp dab chromogenic substrate kit, by Tiangen Biotech (1 mentions)
Pv 6002, by ZSGB-BIO (1 mentions)
Pv 6001, by ZSGB-BIO (1 mentions)
Ab5690, by Abcam (1 mentions)
Af366, by R&D Systems (1 mentions)
Fluorescein labeled secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Ab253297, by Abcam (1 mentions)
Ab197341, by Abcam (1 mentions)
Zm 0038, by ZSGB-BIO (1 mentions)
Zm 0464, by ZSGB-BIO (1 mentions)
5 protocols using zm 0418
1
Immunohistochemical Analysis of CD4+ and CD8+ T Cells
2
Immunohistochemistry and Immunofluorescence Profiling of Kidney Tissues
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Paraffin-embedded kidney tissue sections were used for immunohistochemistry. Briefly, the sections were incubated with primary antibodies to CD3 (1:100, ab5690, Abcam, USA), CD4 (ZM-0418, ZSGB-Bio, China) and CD8 (ZA-0508, ZSGB-Bio, China) overnight at 4 °C and then analyzed with streptavidin peroxidase detection system (Maixin Technology Co., Ltd., China) according to the manufacturer’s protocol.
Immunofluorescence staining was performed to localize the expression of CCL21 and CD3 with tissue sections using anti-CCL21 (1:100, AF366, R&D Systems, USA) and anti-CD3 (1:100, ab5690, Abcam, USA) antibodies in a chamber overnight at 4 °C, followed by incubation with fluorescein-labeled secondary antibodies (Invitrogen, USA) for 1 h. DAPI was used for cell nuclei staining. Immuno-stained samples were visualized using a confocal microscope.
Immunofluorescence staining was performed to localize the expression of CCL21 and CD3 with tissue sections using anti-CCL21 (1:100, AF366, R&D Systems, USA) and anti-CD3 (1:100, ab5690, Abcam, USA) antibodies in a chamber overnight at 4 °C, followed by incubation with fluorescein-labeled secondary antibodies (Invitrogen, USA) for 1 h. DAPI was used for cell nuclei staining. Immuno-stained samples were visualized using a confocal microscope.
3
Immunohistochemical Analysis of CD8 and CD4 T Cells
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The sections of spleen tissue were dewaxed, rehydrated, and then heated by immersing slides in Tris-EDTA buffer (pH 9.0) for 5 min for antigen retrieval. Subsequently, normal goat serum was used to block non-specific binding and 3% H2O2 was applied to suppress endogenous peroxidase activity to reduce background staining. The following antibodies were incubated as the manufacturer’s instructions: rabbit anti-human CD8 Ab (ab93278, abcam) and mouse anti-human CD4 (T Helper/Inducer) monoclonal antibody (mAb) (ZM-0418, ZSGB-BIO) in a humidified chamber overnight at 4°C. After thoroughly washing the corresponding slides for 30 min, horseradish peroxidase labeled-goat anti rabbit IgG Ab and goat anti mouse IgG Ab were added. Finally, staining of the tissue sections was performed with an enhanced HRP-DAB chromogenic substrate kit. The sections were counterstained with immunohistochemical staining and visualized under a light microscope (Nikon, Japan).
4
Immunohistochemical Profiling of Tumor Microenvironment
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All tissue sections were subjected to IHC as previously described [23 (link)]. In brief, paraffin‐embedded sections were cut 4 μm thick, then deparaffinized and rehydrated. Antigenic retrieval was processed with sodium citrate. The sections were then incubated in 3% H2O2 for 10 min, blocked in 1% BSA for 60 min followed by immunostaining using anti‐PD‐L1 mAb (Zsbio, Beijing, China, #ZM‐0381, clone UMAB199, 1 : 100 dilution), anti‐CD4 (Zsbio, #ZM‐0418, clone UMAB64, 1 : 75 dilution) and anti‐CD8 (Zsbio, #ZA‐0508, clone EP334, 1 : 75 dilution). The IHC images were quantified by a number of cells·mm−2. The most and least filtrated regions were defined as regions with maximum and minimum values, respectively, as determined by IHC quantification.
5
Immunohistochemical Analysis of Tumor Immune Markers
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IHC was performed as previously described [28 (link)], and serial sections were incubated with primary antibodies such as anti‐CD161 (67537‐1‐Ig, Proteintech), anti‐LLT1 (ab197341, Abcam), anti‐CD4 (ZM‐0418, ZSGB‐BIO), anti‐CD8 (ZA‐0508, ZSGB‐BIO), anti‐CD19 (ZM‐0038, ZSGB‐BIO), anti‐CD68 (ZM‐0464, ZSGB‐BIO), and anti‐Foxp3 (ab253297, Abcam).
The IHC staining results of CD161 and LLT1 were independently and double‐blindly evaluated by two senior pathologists (Liang Ding and Qingang Hu) who were blinded to the patients' data, and the average values were calculated for further analysis. IHC staining was scored according to the percentage of positive cells and staining intensity. The percentage of stained cells was defined as 0 = 0–5%; 1 = 6–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 75–100%. The staining intensity was defined as follows: 0 = negative staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The IHC score was calculated by multiplying the grade of the staining intensity by that of the staining percentage. High and low expression of LLT1 were defined as the median of IHC scores. The readout score of CD161 lymphocytes was subdivided into values for the tumor center and invasive margin, and high and low expression of CD161 lymphocytes were defined according to the median of the sum of these two values.
The IHC staining results of CD161 and LLT1 were independently and double‐blindly evaluated by two senior pathologists (Liang Ding and Qingang Hu) who were blinded to the patients' data, and the average values were calculated for further analysis. IHC staining was scored according to the percentage of positive cells and staining intensity. The percentage of stained cells was defined as 0 = 0–5%; 1 = 6–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 75–100%. The staining intensity was defined as follows: 0 = negative staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The IHC score was calculated by multiplying the grade of the staining intensity by that of the staining percentage. High and low expression of LLT1 were defined as the median of IHC scores. The readout score of CD161 lymphocytes was subdivided into values for the tumor center and invasive margin, and high and low expression of CD161 lymphocytes were defined according to the median of the sum of these two values.
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