Samples were fixed in 4% paraformaldehyde (PFA) overnight prior to standard processing for paraffin embedding. Serial paraffin sections were cut at 5 μm thickness. Haematoxylin and eosin, Periodic acid-Schiff, picrosirius red and Masson’s trichrome staining were performed on sections using an automated staining system (Tissue-Tek). For immunofluorescence staining, slides were dewaxed using an automated protocol and antigen retrieval was performed using citrate buffer (pH 6.0). Primary antibodies against cytokeratin 5 (CK5; Abcam; ab17130), collagen IV (COL-94; Abcam; ab6311) and laminin (Sigma; L9393) were used. Species-appropriate secondary antibodies conjugated to AlexaFluor dyes (Molecular Probes) were then used. Images were acquired using a Zeiss LSM700 confocal microscope. For immunohistochemistry, slides were dewaxed and antigen retrieved as for immunofluorescence. Primary antibodies against CD45 (Abcam; ab10558), endomucin (V.7C7.1; Abcam; ab106100) and F4/80 (Serotec; MCA497GA) were used. Sections were then incubated with species-appropriate biotinylated F(ab’)2 for 30 min, developed using ABC reagent and superDAB (Dako) and finally counterstained with haematoxylin.
Abc reagent
Manufactured by Agilent Technologies
Sourced in United States
The ABC reagent is a laboratory product manufactured by Agilent Technologies. It is a chemical solution used in various analytical and research applications. The core function of the ABC reagent is to enable specific chemical reactions or processes necessary for the analysis or detection of target analytes. The detailed description and intended use of the ABC reagent will require further information that is not available within the scope of this request.
Automatically generated - may contain errors
Lab products found in correlation
Target retrieval solution, by Agilent Technologies (5 mentions)
3 3 diaminobenzidine (dab), by Merck Group (5 mentions)
Biotinylated anti mouse secondary antibody, by Vector Laboratories (3 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ab17130, by Abcam (1 mentions)
Ab10558, by Abcam (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Superdab, by Agilent Technologies (1 mentions)
Mca497ga, by Bio-Rad (1 mentions)
L9393, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Cox 2, by Abcam (1 mentions)
Hrp labeled polymer anti rabbit antibody, by Agilent Technologies (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Tgf β1, by Abcam (1 mentions)
8 protocols using abc reagent
1
Histological and Immunostaining Analysis of Tissues
2
NUTM1 Protein Immunohistochemical Staining
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Histological specimens were reviewed by a gynecologic pathologist (TM). In addition, immunohistochemical staining for the NUTM1 protein was performed as previously reported.14 , 26 Briefly, after deparaffinization, antigen retrieval was performed with Target Retrieval Solution (10 mM citrate buffer, pH 6.0; Dako) in a microwave for 30 minutes at 96°C. Subsequently, the sections were incubated overnight with primary antibody (C52B1, Cell signaling technology; dilution ratio 1:50) at 4°C. Then, biotinylated anti‐mouse secondary antibodies (Vector Laboratories, Burlingame, CA) were added followed by incubation with ABC reagent (Dako) and 3,3′‐diaminobenzidine (Sigma, St. Louis, MO). Slides were counterstained with hematoxylin. When most of cells were stained, the sample was considered positive. Otherwise, the result was considered negative.
3
Immunohistochemical Analysis of Tumor Samples
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Primary or mouse xenograft tumors were fixed in neutral formalin, embedded in paraffin, and used for staining with hematoxylin and eosin. For immunostaining, the sections were stained following standard IHC methods, as previously described,26 with slight modifications. Briefly, after deparaffinization, antigen retrieval was carried out with Target Retrieval Solution (10 mM citrate buffer, pH 6.0; Dako) in a microwave for 30 min at 96°C. Subsequently, the sections were incubated with primary anti‐SOX2, anti‐p21 (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA), anti‐ p53 or anti‐Ki‐67 antibodies (1:200 dilution; Dako) and biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), followed by incubation with ABC reagent (Dako) and 3,3’‐diaminobenzidine (DAB; Sigma, St. Louis, MO, USA). Slides were counterstained with hematoxylin.
4
Immunohistochemical Analysis of FGFR3 in Xenograft Tumors
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Xenograft tumors were fixed in neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin. All histological specimens were reviewed by a gynecology pathologist (T.M.). In addition, immunohistochemical staining for the FGFR3 protein was performed, as previously reported35 . Briefly, after deparaffinization, antigen retrieval was carried out with Target Retrieval Solution (10 mM citrate buffer, pH 6.0; Dako) in a microwave for 30 min at 96°C. Subsequently, the sections were incubated overnight with primary antibody (sc-13121, Santa Cruz; dilution ratio 1:50) at 4°C and biotinylated anti-mouse secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were added, followed by incubation with ABC reagent (Dako) and 3,3′-diaminobenzidine (Sigma, St. Louis, MO, USA). Slides were counterstained with hematoxylin.
5
Immunohistochemical Analysis of Murine Tissues
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Tissues from animals receiving either MOPCpEF or MOPCSVm cells were snap-frozen in O.C.T. compound and stored at −80 °C. Then, 6 μm tissue sections were air-dried and fixed in acetone, rehydrated in PBS, and blocked with 2% normal goat serum in PBS for nonspecific staining. Primary anti-mouse CD3, CD8, H-2 I-Ad/I-Ed, or CD31 (BD PharMingen, Franklin Lakes, NJ, USA) were applied for 60 min at room temperature. All slides were subsequently washed in PBS and incubated for 30 min with biotinylated secondary Abs (goat anti-rat IgG). Bound Abs were then detected with ABC reagent and Vectastain substrate kit (DAKO, Santa Clara, CA, USA) [54 (link)]. For the hematoxylin and eosin staining, murine tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Then, 4 μm sections were cut and stained with hematoxylin and eosin.
6
Immunohistochemical Staining of FFPE Samples
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Immunohistochemical staining of FFPE samples was performed as described in our previous study.38 Briefly, after deparaffinization, antigen retrieval was carried out with Target Retrieval Solution (10 mM citrate buffer, pH 6.0; Dako) in a microwave for 30 minutes at 96°C. Subsequently, the sections were incubated overnight with primary antibody (Cat#7001; RRID:AB_2206626, Dako; dilution ratio 1:50) at 4°C and biotinylated anti‐mouse secondary antibodies (Vector Laboratories) were added, followed by incubation with ABC reagent (Dako) and 3,3′‐diaminobenzidine (Sigma). Slides were counterstained with hematoxylin. p53 overexpression pattern was defined as strong and diffuse nuclear expression in at least 60% of tumor cells.39 FF samples were used for nonkeratinizing SCC in case 2 because FFPE samples were unavailable. For FF samples, we fixed frozen tissue sections with 4% paraformaldehyde at 4°C for 20 minutes followed by methanol at −20°C for 10 minutes. The immunohistochemical staining protocol after fixation was the same as the protocol for FFPE tissue sections.40
7
Immunohistochemical Analysis of p-ERK Protein
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Immunohistochemical analysis of p‐ERK protein expression was performed using FFPE tissue sections. A monoclonal rabbit anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® antibody (#4370, Cell Signaling Technology) was used as the primary antibody. The FFPE tissue sections (4 µm), which were the serial sections used for RNA‐based in situ detection assays, were cut with a microtome. The sections were stained as previously described.21 Briefly, after deparaffinization, antigen retrieval was carried out with Target Retrieval Solution (10 mM citrate buffer, pH 6.0; Dako) in a microwave for 20 minutes at 98°C. Subsequently, the sections were incubated with the primary antibody (1:700 dilution) overnight and biotinylated secondary antibodies (Vector Laboratories) for 1 hour, followed by incubation with ABC reagent (Dako) and 3,3'‐diaminobenzidine (Sigma‐Aldrich) for 3 minutes. The slides were counterstained with hematoxylin.
8
Immunohistochemical Analysis of Prostate Samples
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