Segments from the ileum of Pou2f3+/−, Pou2f3−/−, Cd300lf+/−, and Cd300lf−/− mice were harvested and fixed in 10% neutral buffered formalin. Tissue samples were processed and paraffin embedded. Afterward, slides were deparaffinized and antigen retrieval was performed using a citrate buffer (DAKO). Tissue samples were blocked for 1 h with 2% normal goat serum in 1X tris-buffered saline with Tween 20 (TBST). Primary antibodies were incubated overnight at 4°C. Tuft cells were detected using a DCLK1 antibody (1:200, Abcam), and epithelial cellular adhesion was shown by using the e-cadherin antibody (1:500, BD Bioscience). For differentiated 3D mouse ileal enteroids were harvested and fixed in 4% paraformaldehyde for ~30 min at RT. Cells were submitted for histology and processed as mentioned above. In addition to DCLK staining, CD300lf antibody (1:200, R&D Systems) was added to check expression and location of the MNoV receptor. The immunofluorescence was detected with Zeiss Axio Imager 2 microscope and photos were processed using the ZenPro program.
Citrate buffer
Manufactured by Agilent Technologies
Sourced in Denmark, United States, France, Canada, Japan
Citrate buffer is a laboratory solution used to maintain a stable pH environment. It is a mixture of citric acid and sodium citrate that can be adjusted to a desired pH range. The core function of citrate buffer is to provide a controlled pH environment for various chemical and biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Fujifilm (10 mentions)
Envision kit, by Agilent Technologies (9 mentions)
Lsm 700, by Zeiss (6 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (6 mentions)
Vectashield mounting medium, by Vector Laboratories (4 mentions)
Hematoxylin, by Merck Group (4 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (4 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Agilent Technologies (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Antibody diluent, by Agilent Technologies (3 mentions)
Dclk1 antibody, by Abcam (2 mentions)
Imager 2 microscope, by Zeiss (2 mentions)
Cd300lf antibody, by R&D Systems (2 mentions)
E cadherin antibody, by BD (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Novared, by Vector Laboratories (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
123 protocols using citrate buffer
1
Tuft Cell and Enteroid Immunohistochemistry
2
Immunohistochemical Analysis of GPA Granulomas
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Paraffin-embedded sections from 13 GPA granulomas (12 in lungs, 1 in kidney) of 10 different patients with GPA and each 5 granulomas from patients with tuberculosis and sarcoidosis were processed. The following primary antibodies were used: rabbit anti-human monoclonal antibody (mAb) CD3 (clone SP7, immunoglobulin G (IgG), 1:200; Thermo Fisher Scientific, Fremont, CA, USA) and mouse anti-human CD56 mAb (clone 1B6, IgG1, 1:10; Leica Biosystems, Newcastle upon Tyne, UK). Isotype- and concentration-matched mouse and rabbit control mAb (Dianova, Hamburg, Germany) served as negative controls. Immunoenzyme staining was performed on 2-μm paraffin-embedded sections of formalin-fixed tissue using automated staining with the DAKO autostainer according to the manufacturer’s instructions (Dako, Glostrup, Denmark). Antigen retrieval for CD3 was achieved by steam-cooking the slides in 1 mM ethylenediaminetetraacetic acid buffer (pH 9; Dako) and for CD56 in 10 mM citrate buffer (pH 6.1; Dako) for 30 minutes. 3,3′-Diaminobenzidine was used as the substrate.
3
Immunofluorescence Staining of Tissue Sections
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Single and double immunofluorescence staining was performed as described by us already elsewhere32 (link),42 (link). In brief, deparaffinized tissue sections were heated in citrate buffer, pH 6 (Dako), for 20 min in a pressure cooker. Sections were then blocked (to avoid non-specific bindings) with ready to use 10% normal goat serum (Life Technologies, MD, USA) for 1 h at room temperature before incubating with primary antibody at 4 °C overnight. After washing in TBS-T for 10 min, the sections were incubated with Alexa conjugated secondary antibody (1 : 500 in TBS-T) at room temperature for 2 h. Sections were washed in TBS-T (2 × 10 min) and stained for 10 min with 0.1% Sudan Black in 80% ethanol to suppress endogenous lipofuscin auto-fluorescence. Finally, the sections were washed for 5 min in TBST and mounted with Vectashield mounting medium (Vector Laboratories) containing DAPI.
4
Immunofluorescence Staining of GALC and OLIG2 in PVWM
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Immunofluorescence staining was performed on sections of the PVWM of Case 2, Case 7, and the adult control brain. Deparaffinized and rehydrated sections were steamed in Citrate buffer (Dako) for 30 min and were blocked with Protein Block plus Serum Free (DAKO) for 1 h. Sections were incubated with anti-GALC antibody (1:1000) and anti-OLIG2 (1:200; ab109186; Abcam, Waltham, MA) antibody diluted in Antibody Diluent with Background-Reducing Components (DAKO) overnight at 4 °C. Sections were washed three times with 1xPBS at room temperature and then incubated with secondary antibodies Alexa Fluor 488 and 568 (1:500, Thermo Fisher Scientific, Inc.) diluted with Antibody Diluent with Background-Reducing Components for 1.5 h at room temperature. Sections were washed three times with 1xPBS at room temperature, incubated with 1% Sudan Black for 2 min, washed with distilled water, and mounted with Vectashield mounting media containing DAPI (Vector Laboratories, Newark, CA).
5
Immunohistochemical Analysis of Tumor Markers
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Formalin-fixed, paraffin-embedded sections were dewaxed. Heat induced epitope retrieval was performed in a water bath containing citrate buffer (Dako North America, Carpinteria, CA, USA), blocked with 1% hydrogen peroxide and then treated for 30 min with CAS-Block (Invitrogen, Carlsbad, CA, USA) before primary antibody incubation. The primary antibodies were rabbit monoclonal anti-Ki-67 (1:200 dilution; Thermo Fisher Scientific) and rabbit monoclonal anti-CD31 (1:75 dilution; Abcam). Staining signals were detected using the Starr Trek Universal horseradish peroxidase (HRP) Detection System (Biocare Medical, Pacheco, CA, USA). Immunohistochemistry slides were scanned with a 3DHITECH PANNORAMIC Midi slide scanner, and images were captured using PANNORAMIC Viewer software (3DHITECH, Budapest, Hungary).
6
Immunohistochemical Analysis of YAP and AREG
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Slides from Tumor paraffin‐embedded blocks were pretreated with 0.01 mol/L citrate buffer (pH 6; Dako) for 20 minutes at 100°C, then immunostainings were performed with automated immunohistochemical stainer (Dako). Slides were successively incubated at room temperature in 3% H2O2 for 5 minutes, then with monoclonal antibody (Table S4 ) diluted at 1:200 for 60 minutes at room temperature. Finally, antibody fixation was revealed by the EnVision+ Dual Link System (Dako).
The staining intensity was recorded by two expert pathologists (MA and CD) blinded to treatment arm. YAP was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong), at ×40 magnification. An overall IHC composite score was calculated from the sum of the staining intensity (0‐3) multiplied by the distribution (0%‐100%) from all parts of the slide, giving a H‐score between 0 and 300. AREG was scored as negative (no signal despite positive internal control) or positive at ×40 magnification.
The staining intensity was recorded by two expert pathologists (MA and CD) blinded to treatment arm. YAP was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong), at ×40 magnification. An overall IHC composite score was calculated from the sum of the staining intensity (0‐3) multiplied by the distribution (0%‐100%) from all parts of the slide, giving a H‐score between 0 and 300. AREG was scored as negative (no signal despite positive internal control) or positive at ×40 magnification.
7
Immunohistochemical Analysis of OLFM4, PCNA, Lysozyme, and BMI1
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Immunohistochemistry (IHC) was performed as previously described [25 (link)]. Briefly, the 4-μm thick sections were deparaffinized in xylene, rehydrated in graded alcohol, and washed with 0.01-M PBS (pH 7.4). After the epitope retrieval with a citrate buffer (pH 6.0; Dako, Carpinteria, CA, USA) and the blocking with a blocking solution (Dako), the tissue sections were incubated with anti-OLFM4 (1:400 dilution, Cell Signalling Technology, Danvers, MA, USA), anti-PCNA (1:100 dilution, Abcam, Cambridge, UK), anti-lysozyme (1:3000 dilution, Abcam), and anti-BMI1 (1:400 dilution, Abcam) antibodies overnight at 4 °C. After washing with PBS, the sections were incubated for 30 min with horseradish peroxidase-conjugated secondary antibodies (Dako) and the antigen–antibody interaction was visualized using the chromogenic substrate 3,3′ diaminobezidine (DAB; Dako).
8
Immunohistochemical Analysis of EMT Markers
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Paraffin-embedded tissues were deparaffinized with xylene. For antigen retrieval, tissues were heated in a microwave oven with citrate buffer (DakoCytomation, Glostrup, Denmark). The sections were treated with 0.2% Triton X-100 for permeabilization, then blocked with 3% BSA in PBS for 1 h. Subsequently, the sections were incubated overnight at 4 °C with anti-USP47, anti-E-cadherin, anti-N-cadherin, anti-Snail, anti-vimentin, and anti-proliferating cell nuclear antigen (PCNA) antibodies and washed with PBS. Incubation with anti-rabbit or anti-mouse secondary antibodies was carried out for 1 h at room temperature. The sections were stained using Prolong Antifade with DAPI (Invitrogen).
9
Immunofluorescence Staining of Cultured Cells and Muscle Tissue
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Following fixation (4% paraformaldehyde [PFA], 10 min), cultured cells were washed with PBS, permeabilized for 10 min in 0.1% Triton X-100 (Sigma), and blocked with a solution containing 5% BSA (Sigma) and 0.05% Triton X-100 for 1 h at room temperature. Cells were incubated with primary antibodies overnight at 4°C, and then with appropriate coupled secondary antibodies for 1 h at room temperature.
Muscles were dissected and snap frozen in liquid nitrogen-cooled isopentane. Muscle sections (10 μm) were fixed with 4% PFA for 20 min, permeabilized in cold methanol for 6 min, and boiled in citrate buffer (Dako) for epitope retrieval. The blocking was performed for 2 h at room temperature with BSA 5% and then for 30 min with anti-mouse IgG Fab fragment (Jackson Laboratories). A detailed list of the antibodies used is provided inTable S4 .
Muscles were dissected and snap frozen in liquid nitrogen-cooled isopentane. Muscle sections (10 μm) were fixed with 4% PFA for 20 min, permeabilized in cold methanol for 6 min, and boiled in citrate buffer (Dako) for epitope retrieval. The blocking was performed for 2 h at room temperature with BSA 5% and then for 30 min with anti-mouse IgG Fab fragment (Jackson Laboratories). A detailed list of the antibodies used is provided in
10
Immunohistochemical Analysis of SAA1 and CRP
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Tissue blocks from the subpleural parenchyma (paying attention to avoid areas involved by the tumor) were fixed in 10% formalin and embedded in paraffin. Five-μm-thick sections were prepared for immunohistochemical analysis. Mouse monoclonal antibodies against SAA1 (Novus Biologicals, Cambridge, UK) and CRP (Abcam, Cambridge UK) were used. Antigen retrieval was achieved by microwave heat treatment in citrate buffer (Dako, Glostrup, Denmark) at 98°C for 15 mins. The bound antibody was developed with diaminobenzidine using a Dako Envision staining kit (K4065) according to manufacturer’s recommendations. Stained sections were observed under light microscope by two independent observers. All immunohistochemical studies included appropriate standard quality controls. As a negative control, immunohistochemistry was performed using secondary antibodies without the primary antibody.
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