Whole depth rumen wall tissue and enzymatically separated epithelium and LP were embedded in Tissue-Tek® O.C.T compound (Sakura, CA, USA), snap frozen in liquid nitrogen and cryostat (cryotome SME, Life sciences, Cheshire, UK) sectioned (10 μm). Sections were stained in Wiegert’s iron haematoxylin solution (Sigma, MO, USA) for 6–8 min, and then rinsed in tap water to clear unbound stain. Sections were then immersed in Gomori trichrome stain (Sigma, MO, USA) for 10 min, and the colour differentiated with 0.2% acetic acid. Sections were then dehydrated in ascending ethanol 95–100% for 2 × 5 min each change of solution. The sections were then cleared with xylene substitute (Sigma, MO, USA) for 3 × 5 min. A coverslip was mounted with DPX solution (Sigma, MO, USA), and air dried overnight.
Xylene substitute
Manufactured by Merck Group
Sourced in United States, United Kingdom
Xylene substitute is a laboratory reagent used as an alternative to xylene in various applications. It serves as a clearing agent and a solvent in histological and cytological procedures. The product provides a safe and effective solution for applications where xylene is commonly used.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (12 mentions)
Hoechst dye, by Merck Group (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Hoechst 34580, by Thermo Fisher Scientific (2 mentions)
Haematoxylin, by Merck Group (2 mentions)
Eclipse e1000, by Nikon (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Uea1 fitc conjugated lectin, by Vector Laboratories (2 mentions)
Rabbit anti o152 primary antibody, by Denka Seiken (2 mentions)
Goat anti rabbit alexa 488 conjugated secondary anti bodies, by Thermo Fisher Scientific (2 mentions)
Hoechst dna dye, by Merck Group (2 mentions)
Uea1 dylight647 conjugated lectin, by Vector Laboratories (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Dpx solution, by Merck Group (1 mentions)
Wiegert s iron haematoxylin solution, by Merck Group (1 mentions)
Ab46512, by Abcam (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Bloxall reagent, by Vector Laboratories (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Novared hrp substrate, by Vector Laboratories (1 mentions)
18 protocols using xylene substitute
1
Histological Analysis of Rumen Wall Tissue
2
Chromogenic In Situ Hybridization for EcPV2 Oncogenes
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To assess tissue and derived raft sections for EcPV2 oncogene transcription, RISH was carried out on three representative FFPE tissue and raft sections as described previously [28 (link)]. In brief, a multiple-probe chromogenic RISH method (RNAscope) designed to hybridize to EcPV2 E6/E7 [29 (link)] was run on five replicates per lesion or raft using an RNAscope 2.5 HD Detection Kit RED (ACDbio; Newark, CA, USA) according to the manufacturer’s instructions with minor modifications. Instead of xylene, Xylene Substitute (Sigma-Aldrich) was used; to expose nucleic acids, slides were placed into boiling 1× target retrieval solution for 15 min, and counterstaining was performed in 20% instead of 50% hematoxylin.
3
Immunohistochemical Staining of Colon Tissue
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Distal colon tissue containing a fecal pellet was fixed by submersion in methanol-Carnoy solution for at least 24 h. Fixed tissue was paraffin embedded and cut into 5-µm sections. Tissue sections were deparaffinized by sequential washing in Xylene substitute (20 min at 60°C; Sigma-Aldrich) and 100% (5 min), 95% (5 min), 70% (5 min), and 30% (5 min) ethanol. Antigen retrieval was performed by immersion in 10 mM citrate buffer (95°C, 30 min). Sections were washed in PBS, permeabilized for 5 min using 0.1% vol/vol Triton X-100 (Sigma-Aldrich), and blocked using 5% vol/vol FCS. Sections were incubated overnight at 4°C with primary antibody solutions to stain Muc2 (Muc2C3, 1:500; Johansson et al., 2008 (link)), Apo-Muc2 (PH497, 1:1,000; Hansson et al., 1994 (link)), Clca1 (ab46512, 1:2,000; Abcam), Zg16 (anti-ZG16, 1:600; Rodríguez-Piñeiro et al., 2013 (link)), and Agr2 (H00010551-M01, 1:2,000; Abnova). Sections were washed in PBS, and primary antibodies were detected by incubation with goat anti-rabbit Alexa Fluor 488– or Alexa Fluor 555–conjugated secondary antibodies (1:2,000; Thermo Fisher) for 2 h at room temperature. Finally, slides were rinsed in double-distilled water and counterstained with Hoechst dye (5 µg/ml; Sigma-Aldrich) for 5 min. Stained slides were subsequently imaged using an LSM700 confocal microscope (Zeiss).
4
Fluorescent In Situ Hybridization for Bacterial 16S Identification
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FISH staining for bacterial 16S was performed using the tissue sections described above. Sections were deparaffinized by sequential washing in Xylene substitute (20 min at 60°C; Sigma-Aldrich), 100% ethanol (5 min), and 95% ethanol (5 min). Slides were air dried and flooded with hybridization buffer (40% vol/vol formamide, 0.1% wt/vol SDS, 0.9 M NaCl, and 20 mM Tris, pH 7.4) supplemented with Alexa Fluor 555–labeled universal bacterial FISH probe EUB338 (1 mM). Slides were incubated at 37°C overnight in humidity chambers containing 40% vol/vol formamide solution, subsequently submerged in wash buffer (0.9 M NaCl and 25 mM Tris, pH 7.4), and incubated at 50°C for 20 min. Finally, slides were rinsed in double-distilled water and counterstained with Hoechst dye (5 µg/ml; Sigma-Aldrich) for 5 min. Stained slides were imaged using an LSM700 confocal microscope (Zeiss).
5
Histological Analysis of Cellular Infiltration
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To analyze the cellular infiltration, 5-μM-thick sections were cut from paraffin-embedded tissue blocks and placed on charged microscope slides for staining. Sections were deparaffinized, rehydrated, and subjected to routine hematoxylin (Sigma) and eosin (Sigma) staining. Representative images were acquired using a Zeiss AxioImager microscope. To determine the phenotype of the cells at the injection site, 5-μM sections from paraffin-embedded tissues were cut, prepared, and blocked as described above. Endogenous peroxidase/phosphatase activity was blocked using Bloxall reagent (Vector Labs). Anti-F4/80 (6640; Abcam) and anti-CD3 (RM-9107; Thermo Scientific) as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Peroxidase activity was visualized using NovaRed HRP substrate (Vector Labs). Slides were then counterstained with hematoxylin, dehydrated with ethanol, and cleared with xylene substitute (Sigma). Coverslips were mounted with Organo/Limonene mounting medium (Sigma). Representative images were acquired using a Zeiss AxioImager microscope.
6
Immunohistochemical Analysis of Pig Airway Mucins
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Pig airway tissue was fixed in 4% formalin, embedded in paraffin, and cut in 4-μm-thick sections that were dewaxed using xylene substitute (Sigma) and hydrated. For immunohistochemistry, antigen retrieval was performed by microwave heating in 0.01 m citric buffer (pH 6). Sections were blocked for 60 min with Tris-buffered saline (TBS) with 3% donkey serum and 0.1% Triton X-l00. The primary antibody MUC5B (a kind gift from M. Kesimer, University of South Carolina, Chapel Hill, SC) in blocking solution was incubated overnight at 4 °C and washed in TBS three times for 5 min before adding donkey anti-rabbit Alexa 555 (Thermo Fisher Scientific, Waltham, MA). Nuclei were counterstained with Hoechst 34580 (Thermo Fisher Scientific). Slides were washed in TBS three times for 5 min and mounted with Prolong Gold mounting medium (Thermo Fisher Scientific). Images were acquired with an upright LSM 700 Axio Examiner 2.1 confocal imaging system (Carl Zeiss, Oberkochen, Germany).
7
Immunofluorescent Staining of Colon Tissue
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Human colonic sigmoid biopsies and mouse distal colons were immediately fixed in methanol-Carnoy’s fixative for 48 h with 100% methanol used for long-term storage. The samples were paraffin-embedded and cut into 4-μm section (1 (link), 44 ). Slides were deparaffinized in Xylene substitute (Sigma) and stepwise hydrated. The mouse sections were subjected to antigen retrieval in 10 mM citric acid buffer, pH 6, at 100 °C for 30 min. The samples were blocked using 2% BSA in PBS, and primary antibodies were incubated overnight at 4 °C. For mouse tissue, the αMUC2C3, αFCGBP-D13, and αFCGBP-N sera were used at 1:2000 dilutions and at 1:4000 for human samples. FCGBP pre-serums (before immunization) for αFCGBP-N and αFCGBP-D13 were used as negative controls. Goat anti-rabbit-Alexa 555 (Thermo Fisher Scientific) diluted 1:2000 was used as the secondary antibody, and DNA was counterstained with Hoechst-34580 (1 μg/μl, Thermo Fisher Scientific). The sections were mounted in ProLong Gold antifade (Thermo Fisher Scientific). Images were acquired using a Nikon Eclipse E1000 fluorescence microscope with a 20× 0.50 NA Plan Fluor DIC objective (Nikon). The 64-bit NIS-Element D (version 4.20.01, Nikon) and Imaris Viewer (version 9.5.1, Imaris) were used for image processing.
8
Histological Tissue Preparation Protocol
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Alginic acid, CaCl2, casein solution, DPX mountant, eosin, EDTA, EtOH, haematoxylin, NaCl, paraformaldehyde (PFA), polyvinylpyrrolidone (PVP), sodium citrate, Triton™ X-100 and xylene substitute were purchased from Sigma-Aldrich (Gillingham, UK). Hydroxypropyl-methylcellulose (HPMC) was purchased from Alfa Aesar (Thermo Fisher Scientific, Heysham, UK).
9
Immunohistochemical Detection of TRPM4
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Tissue sections were incubated at 60°C for 10 min to facilitate tissue adherence onto the slides before deparaffinization in two changes of xylene substitute (Sigma-Aldrich Co., St Louis, MO, USA) each for 15 min. This was followed by serial rehydration in graded ethanol (GmbH, Hamburg, Germany) from 100% ethanol followed by 70%, 50% and 30% ethanol, and finally in distilled water. Heat-mediated antigen retrieval was conducted in Tris-EDTA buffer (pH 9.0) using a microwave pressure cooker for 10 min followed by incubation with a mouse anti-TRPM4 monoclonal antibody (clone 10H5; Abcam, Cambridge, UK) at 1:500 dilution (1.536 μg/ml) for one hour at room temperature. Binding of the anti-TRPM4 antibody was detected using HRP-conjugated secondary anti-mouse/rabbit antibody from EnVisionTM detection system (DakoCytomation, Carpinteria, CA, USA) for 30 min and developed with DAB as the chromogen for 5 min. The sections were counterstained with fresh Gill No. 2 hematoxylin solution (Sigma Aldrich) for 10 sec and mounted with the VectaMountTM (Vector Labs, Burlingame, California) non-aqueous mounting medium.
10
Immunofluorescent MUC2 Staining in Mice
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Whole tissues from CF and WT mice were fixed in Carnoy's fixative (60% dry methanol, 30% chloroform and 10% glacial acetic acid), embedded in paraffin and cut in 4-μm thick sections. The sections were dewaxed using Xylene substitute (Sigma, St. Louis, MO, USA) and hydrated. Antigen retrieval was carried out by microwave heating in 0.01 mol/L citric buffer pH 6, and the sections were stained with custom-made anti-MUC2C3 antiserum (1 : 500) and goat anti-rabbit Alexa 488 secondary antibody (Invitrogen).27 (link) DNA was stained by TO-PRO-3 Iodide (1 μmol/L, 642/661; Invitrogen). Images were acquired using a fluorescence microscope, Eclipse E1000 with a Plan-Fluor 40×/0.75 DIC objective (Nikon, Amstelveen, the Netherlands). The pictures were processed uniformly using adobe photoshop (Adobe, San Jose, CA, USA).
Data are presented as mean ± standard error of the mean (SEM) for n animals. The Mann–Whitney test was used to test differences between two groups and the Kruskal–Wallis with Dunn's multiple comparisons test was used to test differences between multiple groups. Statistical significance was accepted when P < 0.05.
Data are presented as mean ± standard error of the mean (SEM) for n animals. The Mann–Whitney test was used to test differences between two groups and the Kruskal–Wallis with Dunn's multiple comparisons test was used to test differences between multiple groups. Statistical significance was accepted when P < 0.05.
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