Treg cell infiltration in local tissue was quantified by Immunohistochemistry. Tumor specimens and normal nasal mucosa were fixed, embedded, and sectioned at 5μm thickness. 15 samples each from SSCC, NIP and normal control subjects were assessed by fluorescent immunohistochemistry, using rabbit-anti-CD4 (staining red) and mouse-anti-Foxp3 (staining green) (both from abcam, UK) to identify Treg cells. The stained sections were observed by two independent physicians, blinded to the clinical diagnosis of the patients, using a fluorescence microscope. All samples were assessed at x400 magnification and double-stained cells CD4+Foxp3+ Treg cells were counted in10 randomly selected fields per sample. The results were expressed as the mean number of CD4+Foxp3+ Treg cells in 10 fields.
Mouse anti foxp3
Manufactured by Abcam
Sourced in United States
Mouse-anti-Foxp3 is a primary antibody that detects the Foxp3 transcription factor. Foxp3 is a key regulator of T-cell development and function. This antibody can be used for applications such as flow cytometry and immunohistochemistry to identify and study Foxp3-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated second antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti cd3, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Rabbit anti cd8, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Rabbit anti rorγt, by Abcam (1 mentions)
Rabbit anti il 17, by Abcam (1 mentions)
Mouse anti cd68, by Abcam (1 mentions)
Goat anti il 17, by R&D Systems (1 mentions)
5 protocols using mouse anti foxp3
1
Quantification of Treg Cells in Nasal Mucosa
2
Immunohistochemical Profiling of Immune Cells
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Pathological diagnoses were confirmed by standard H&E staining. Paraffin-embedded, 4-lm-thick sections were selected for IHC analysis. Sections were dewaxed and then subjected to heat-induced epitope retrieval with a preheated epitope retrieval solution (10 mM citrate buffer, pH 6.0). Next, endogenous peroxidase activity was blocked, and the sections were incubated overnight with one of the following primary mAbs: mouse anti-CD3, mouse anti-CD4, mouse anti-CD20, mouse anti-CD57, mouse anti-CD68, rabbit anti-CD8 (all were working solutions; from Zhongshan Golden Bridge Biotechnology, Beijing, China), or mouse anti-FOXP3 (1:400; Abcam, Cambridge, UK, ab20034). After incubation with the HRP-conjugated second antibody (Invitrogen, Carlsbad, CA, USA, 11) and development with diaminobenzidine, the sections were counterstained with haematoxylin.
Negative control staining was performed with cold PBS in place of the primary antibody.
Negative control staining was performed with cold PBS in place of the primary antibody.
3
Quantifying pDCs and Tregs by IHC
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Blood dendritic cell antigen-2 (BDCA2) and Foxp3 were selected as cell markers of pDCs and Tregs, respectively. The expression of these immunosuppressive proteins in tissues was evaluated via immunohistochemical analysis. Serial 5-μm frozen sections were used in this study. The details of the IHC procedure were performed as described in our previous studies (29 (link), 34 (link)). Commercially available primary antibodies were used according to the manufacturer's instructions (mouse anti-BDCA2, 1:800 dilution, MACS, CA; mouse anti-Foxp3, 1:200 dilution, Abcam, Cambridge, MA, USA). Negative control staining was carried out with cold PBS in place of primary antibody. Numbers of BDCA2+pDCs and Foxp3+Tregs in each of 5 high-power fields (magnification 400 ×) were counted by two blinded, independent researchers using a microscope (objective lens, 40 ×; ocular lens, 10 ×; BX41 microscope, Olympus, Japan).
4
Immunohistochemical Analysis of FOXP3, IL-17, and RORγt
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The Envision method was used for immunohistochemistry. Sections were deparaffinized, rehydrated, antigen-retrieved with sodium citrate buffer, and blocked with 20% normal goat serum in a humidified chamber at 37°C for 30 min and then incubated with primary antibodies (mouse anti FOXP3, 1 : 100, Abcam; rabbit anti-IL-17 and rabbit anti-RORγt, 1 : 100, Abcam) at 4°C overnight. Sections were then incubated with HRP-labeled secondary antibodies (rabbit anti-mouse IgG, HRP Conjugated for FOXP3, goat anti-rabbit IgG, HRP Conjugated for IL-17 and RORγt) at 37°C for 60 min, followed by DAB for color development. Sections were then counterstained with hematoxylin, differentiated in a 0.1% hydrochloric acid-ethanol solution, and mounted with a neutral resin. Three microscopic fields were randomly chosen from each slide and analyzed with the IPP image analysis system to obtain integrated optic density (IOD) for the positive staining.
5
Immunohistochemical Analysis of Immune Markers
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Immunohistochemistry was performed as described in our previous study [20] (link). Primary antibodies were goat anti-IL-17 (R&D Systems, UK), mouse anti-CD66b (BD Pharmingen, USA), mouse anti-CD68 (Abcam, USA), mouse anti-FoxP3 (Abcam, USA), and mouse anti-CD34 (Beijing Zhongshan Golden Bridge Biotech, China). For the negative control, the primary antibody was replaced with phosphate-buffered saline (PBS).
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