For paraffin embedding, adrenal glands were dissected and the surrounding fat was removed and fixed in 10% neutral buffered formalin (Sigma-Aldrich, HT501128) overnight at room temperature. Tissue was embedded manually over 3 days. All washes were carried out for 1 hour at room temperature unless indicated. Day 1: three times PBS, 25% EtOH, 50% EtOH, 70% EtOH, and 70% EtOH overnight at 4°C. Day 2: 80% EtOH, 90% EtOH, 95% EtOH, and 100% EtOH overnight at 4°C. Day 3: 100% EtOH, Neoclear (Sigma-Aldrich, 109843) I for 10 min at room temperature; Neoclear II for 10 min at 60°C; Neoclear: paraffin 1:1 for 15 min at 60°C, paraffin I for 1 hour at 60°C, paraffin II for 1 hour at 60°C, and paraffin III for 1 hour at 60°C. Samples were sectioned at 5 μm. RNAscope was carried out on paraffin-embedded sections with the RNAscope 2.5 HD Kit-BROWN (ACD bio 322300) assay following the manufacturer’s protocols, with “Standard” timings for retrieval and protease treatment. The following probes were used (all ACD bio): Mm-Abcb1b (422191), Mm-Sbsn (564441), Mm-Srd5a2 (431361), Hs-ABCB1 (401191), and Hs-SBSN (447411). Positive control Mm-Ppib (313911), Hs-UBC (310041), and negative control dapB (310043) were also used. Nuclei were stained with Vector Hematoxylin QS (Vector Laboratories, H-3404), and slides were mounted in VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000).
Neo clear
Manufactured by Merck Group
Sourced in Germany, United States
Neo-Clear is a laboratory equipment designed for the purification and analysis of various biological samples. It utilizes advanced filtration and detection technologies to provide precise and reliable results. The core function of Neo-Clear is to facilitate the separation and isolation of target analytes from complex mixtures, enabling researchers to obtain high-purity samples for further investigation.
Automatically generated - may contain errors
Lab products found in correlation
Neo mount, by Merck Group (7 mentions)
Dfc295, by Leica camera (3 mentions)
Dm2000, by Leica camera (3 mentions)
Paraffin, by Merck Group (3 mentions)
Buffered formalin, by Merck Group (3 mentions)
Mayer s haemalaun, by Merck Group (2 mentions)
Kaiser s glycerin gelatine, by Merck Group (2 mentions)
Ht110216, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Fast green, by Thermo Fisher Scientific (1 mentions)
Direct red 80, by Merck Group (1 mentions)
Picric acid, by Merck Group (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Mouse monoclonal antibody, by Merck Group (1 mentions)
Goat polyclonal antibody, by R&D Systems (1 mentions)
Anti st2, by Merck Group (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Diaminobenzidine, by Merck Group (1 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
39 protocols using neo clear
1
Paraffin Embedding of Adrenal Glands
2
Histological Analysis of Human Gingival Tissue
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Human gingival tissue was freshly collected and fixed overnight in 4% neutral buffered formalin. Then, tissue underwent three 5 min washes in PBS at room temperature followed by dehydration washes in increasing ethanol concentrations. After dehydration, tissue was processed using a Leica ASP300 Tissue Processing for one hour. Tissues were then embedded in paraffin. Serial sections (12 µm thick) were cut for haematoxylin and eosin (H and E) and immunohistochemistry (IHC) staining.
H and E was carried out for each patient sample using an Automated Slide Stainer. Slides were dewaxed by immersion in Neo-Clear (Merck Millipore), twice for 10 min. Tissue was then rehydrated by decreasing volumes of ethanol in deionised H20 (100, 90, 70, 50%) for two minutes in each step and rinsed in deionised H20 for 2 min. Samples were then stained in Ehrlich’s Haematoxylin (Solmedia) for 10 min followed by a 10 min rinse under running water and then a two-minute rinse in deionised H20. Tissue was then stained in 0.5% Eosin Y (Sigma-Aldrich) for 5 min and washed twice in deionised H20. Samples were dehydrated in increasing IMS in deionised H20 concentration steps (70, 90, 100, 100%) for two minutes each. Slides were immersed in Neo-Clear three times for 5 min and then mounted using Neo-mount mounting medium (Merck Millipore), coverslipped and left to dry overnight in at 42°C.
H and E was carried out for each patient sample using an Automated Slide Stainer. Slides were dewaxed by immersion in Neo-Clear (Merck Millipore), twice for 10 min. Tissue was then rehydrated by decreasing volumes of ethanol in deionised H20 (100, 90, 70, 50%) for two minutes in each step and rinsed in deionised H20 for 2 min. Samples were then stained in Ehrlich’s Haematoxylin (Solmedia) for 10 min followed by a 10 min rinse under running water and then a two-minute rinse in deionised H20. Tissue was then stained in 0.5% Eosin Y (Sigma-Aldrich) for 5 min and washed twice in deionised H20. Samples were dehydrated in increasing IMS in deionised H20 concentration steps (70, 90, 100, 100%) for two minutes each. Slides were immersed in Neo-Clear three times for 5 min and then mounted using Neo-mount mounting medium (Merck Millipore), coverslipped and left to dry overnight in at 42°C.
3
Quantifying Myocardial Fibrosis via SRFG Stain
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Fibrosis was assessed in myocardial sections by Sirius Red Fast Green (SRFG) stain, as previously reported with modification.5 Briefly, 10 µm thick mid-chamber coronal sections were fixed in ice cold acetone for 3 hours. Slides were rehydrated in 70% ethanol and rinsed with tap water. Sections were stained in 0.1% Direct Red 80 (Sigma #365548) and 0.1% Fast Green (Fisher #F88–10) in 1.3% Picric Acid (Sigma #P6744) for 25 min. and rinsed with tap water. Slides were sequentially dehydrated in 70% ethanol followed by 100% ethanol. Sections were cleared with Neo-Clear and mounted with NeoMount (Millipore). Fibrosis was confirmed with Masson’s Trichrome stains as previously described.(37 (link))
4
Histological Analysis of Embryos
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For histological analysis, embryos were fixed overnight at 4°C in 4% paraformaldehyde (PFA) in PBS, pH 7.4. Fixed embryos were gradually dehydrated with 70%, 90%, and 100% EtOH, followed by immersion in Neo-Clear (Millipore, 65351). Tissues were embedded in paraffin wax at 58°C and sectioned transversely or sagittally with 7 μm thickness. Immunostaining of paraffin sections and whole-mount immunohistochemical staining of embryos were performed as described [32 (link)]. Paraffin-embedded slides were freshly treated with Neo-Clear twice for 10 min each, followed by gradual rehydration in EtOH (100%, 90%, 80%, and 70%; 6 min each) and water for 20 min. For immunohistochemistry, the slides were treated with blocking solution (5% normal goat serum and 0.2% Triton X-100 in PBS) for 1 h and incubated with primary antibodies and subsequently secondary antibodies. Confocal images were taken with a Zeiss LSM 700 laser scanning confocal microscope equipped with Zeiss C-Apochromat 60x (1.2 NA) and 40x (1.2 NA) water immersion lens and analyzed using ZEN (black edition) 2012 SP5 software (Zeiss). Using the ZEN software, z-stacks of images covering the entire cell thickness were acquired and projected maximally. Image processing and annotation was done with Adobe Photoshop, Adobe Illustrator and Fiji software [60 (link)].
5
Immunofluorescent Analysis of ST2/E-cadherin and IL-33/α-SMA
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The co-expression of ST2/E-cadherin and IL-33/α-SMA in paraffin histological partitions from the healthy and tumor samples was evaluated using immunofluorescence. In a nutshell, Merck KGaA NeoClear was used to deparaffinize the sections, and then a variety of alcohols, ranging from 99% to 65% ethanol, was used to rehydrate them. The antigenic healing was performed using sodium citrate with pH = 6 for ST2/Ecad and EDTA with pH = 7.5 for IL-33/-SMA. In 2x PBS containing 2% normal donkey serum, 4% bovine serum albumin (BSA), and 150 mM glycine, the sections were then treated (for non-specific protein plugging and autofluorescence, respectively). The compartments were incubated for an hour at 37˚C with the primary antibodies including anti-α-SMA (1/500), anti-IL-33 (1/500), anti-ST2 (1/1,000), monoclonal mouse antibody (Sigma-Aldrich), and polyclonal goat antibody (R and D Systems). After tissue slices had been rinsed in PBS for an hour at 37˚ C, secondary antibodies were added. Hoechst 33,342 (1/1,000) was operated to create nuclear counterstain. Lastly, a coverslip and mounting solution were used to cover the slides (Dako). The confocal microscope was utilized to view the slides at ×60 and ×20 magnifications.
6
Quantification of Ki67-positive Cells in Dentate Gyrus
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Briefly, brain sections stored in CPS were transferred into PBS and washed. Endogenous peroxidase activity was blocked by adding 0.6% hydrogen peroxide (H2O2; Merck Millipore) for 30 min and sections were then rinsed with 0.9% NaCl. Protein-binding sites were blocked with a blocking solution (10% donkey serum, 0.2% Triton X-100 in PBS) for 1 h. Ki67 staining was performed with the Ki67 primary (rabbit anti-Ki67, 1:500; Novocastra) and donkey anti-rabbit-biotin secondary antibodies (1:1000; Jackson Immunoresearch Laboratories). Detection was performed using the Vectastain ABC-Elite reagent (Vector Laboratories) with diaminobenzidine (Sigma-Aldrich) and 0.04% NiCl as the chromogen. Sections were mounted onto gelatin-coated glass slides, dried, cleared with Neoclear (Merck) and coverslipped using Neo-mount (Merck). Every sixth section (240 μm apart) was counted in the complete ventral dorsal extent of the dentate gyrus, at 40x magnification using a standard brightfield microscope. Results were multiplied by 6 in order to obtain the total number of positive cells within the DG region of each brain.
7
Quantitative Analysis of Angiogenesis and Proliferation Markers
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Microscope slides with 3 μm sections from paraffin-embedded tissue were dewaxed and rehydrated following standard procedures (preheating at 60°C, xylene substitute [Neo-Clear, Merck KgaA, Darmstadt, Germany] graded series of ethanol (100%, 96%, 80% and 70%), followed by double distilled water). After antigen demasking (microwave irradiation at 600 W, 0.1 M citrate buffer pH 6.0) and overnight incubation with the primary antibodies (anti-ß3-integrin antibody Abcam ab179473, 1:500, anti-CD31 antibody Abcam ab28364 1:50, anti-Ki-67 antibody SP6 Abcam ab16667 1:100 [all Cambridge, United Kingdom]) at 4°C, tissue samples were further processed using the EnVision+ System HRP (DAB or AEC) (DAKO Diagnostika, Hamburg, Germany) kit according to the manufacturer´s instructions. Slides were counterstained with Mayer´s Haemalaun (Merck KgaA, Darmstadt, Germany) and covered with Kaiser´s Glycerin Gelatine (Merck KgaA, Darmstadt, Germany). The optical density of ß3-integrin expression was measured in ten random fields at 200x magnification using ImageJ (“Fiji” version, www.fiji.sc ). CD31-positive microvessels and Ki-67-positive nuclei were quantified in ten random high-power fields at 200x magnification.
8
Immunohistochemical Staining for L1CAM in Tissue Sections
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Tissue specimens were sectioned at 4-µm thickness, deparaffinized in Neo-clear (Merck KGaA) for 25 min, and rehydrated by passage through a graded alcohol series for 25 min. Heat-induced epitope retrieval was performed in Target Retrieval Solution pH 9 (Dako; Agilent Technologies, Inc.) for 10 min using a microwave. Endogenous peroxidase was inactivated by incubation in 3% H2O2 solution for 10 min. Immunohistochemical staining for L1CAM was performed using a mouse monoclonal anti-human L1CAM antibody (1:100; clone 14.10; cat. no. 826701; BioLegend Inc.) for 1 h at room temperature (RT). Slides were rinsed in DAKO wash buffer and then incubated for 30 min with peroxidase-labeled polymer conjugated to anti-mouse immunoglobulins (EnVision Detection System; Dako; Agilent Technologies, Inc.). The chromogenic reaction was carried out with DAB chromogen. All sections were counterstained with Mayer's hematoxylin for 1 min at RT.
9
Histological Analysis of DMBA-Induced Oral Carcinogenesis
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Thirteen weeks after the first DMBA exposure, animals were euthanized, right buccal pouches were procured, and tumors were resected. Samples were fixed in 10% buffered formalin (Merck, USA), embedded in paraffin (Merck), and sectioned. Tissue sections of 4 μm were deparaffinized with Neoclear (Merck), rehydrated with graded alcohols, stained with hematoxylin–eosin (Merck), and visualized with a light microscope (DM2000; Leica, Germany). Images were captured with a digital camera (DFC295; Leica). Samples were classified as hyperplasia, dysplasia, papilloma, or carcinoma as described previously [18 (link), 19 (link)].
Histological analyses were performed blind by three independent observers; one of them is a pathologist expert in oral diseases.
Histological analyses were performed blind by three independent observers; one of them is a pathologist expert in oral diseases.
10
Histological Analysis of Hamster OSCC
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After five weeks of treatment, the hamsters were euthanized with an overdose of 20 mg/Kg Xylazine and 20 mg/Kg Ketamine by intraperitoneal injection.
Buccal pouches were procured, and tumors were resected for standard histological analyses. In brief, tumor specimens were fixed in 10% buffered formalin (Merck) and embedded in paraffin (Merck). Four μm sections were cut from each embedded sample, deparaffinized with Neoclear (Merck), and rehydrated with graded alcohols. Haematoxylin and Eosin stains (H&E) (Merck) were applied to the sections, which were imaged using a light microscope (DM2000, Leica) with a digital camera (DFC295, Leica). For OSCC assessment, the carcinoma stage was defined as previously described [25 (link)]. The histological evaluation was performed by two independent observers blinded to outcome.
Buccal pouches were procured, and tumors were resected for standard histological analyses. In brief, tumor specimens were fixed in 10% buffered formalin (Merck) and embedded in paraffin (Merck). Four μm sections were cut from each embedded sample, deparaffinized with Neoclear (Merck), and rehydrated with graded alcohols. Haematoxylin and Eosin stains (H&E) (Merck) were applied to the sections, which were imaged using a light microscope (DM2000, Leica) with a digital camera (DFC295, Leica). For OSCC assessment, the carcinoma stage was defined as previously described [25 (link)]. The histological evaluation was performed by two independent observers blinded to outcome.
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