Mice were sacrificed and the subcutaneous tumors were dissected. Tumors were fixed in 10% formalin (Fisher, cat. SF98–4) at room temperature overnight and consequentially dehydrated in 70% ethanol. Samples were mounted in paraffin blocks and sectioned at 5 μm of thickness. Haematoxylin and eosin staining was performed in Lecia Autostainer XL. Briefly, slides were baked at 60°C for 15 minutes, then treated with Xylene Substitute (Citrisolv) (Fisher chemical cat. x3p-1gal) then rehydrated and stained with Hematoxylin, followed with washes with running water. An incubation with Acid Alcohol (Sigma, cat. A3429) was performed, followed with washes with running water. The samples were then stained with eosin (Thermo Scientific) then dehydrated with ethanol followed by Xylene Substitute (Citrisolv). Samples were mounted with Permount (Fisher Scientific, cat. SP15–500). Slides were submitted for pathologic evaluation. Pathologist was blinded to sample origin (i.e., experimental versus control)
Xylene substitute
Manufactured by Thermo Fisher Scientific
Xylene Substitute is a non-toxic, environmentally-friendly alternative to xylene for use in histology and cytology laboratories. It is designed to effectively clear paraffin from tissue sections, enabling subsequent staining procedures. The product provides a safe and efficient substitute for traditional xylene-based clearing agents.
Automatically generated - may contain errors
Lab products found in correlation
Acid alcohol, by Merck Group (7 mentions)
Permount, by Thermo Fisher Scientific (6 mentions)
Autostainer xl, by Leica (4 mentions)
Real detection system, by Agilent Technologies (3 mentions)
Dab kit, by Thermo Fisher Scientific (3 mentions)
Universal blocking reagent, by BioGenex (3 mentions)
Permount solution, by Thermo Fisher Scientific (2 mentions)
Sodium citrate, by Merck Group (2 mentions)
Donatello, by Diapath (2 mentions)
0.1 m sodium citrate, by Merck Group (1 mentions)
Mounting medium, by Thermo Fisher Scientific (1 mentions)
Zen lite software, by Zeiss (1 mentions)
Mayer s hematoxylin solution, by Merck Group (1 mentions)
Axio imager d2, by Zeiss (1 mentions)
Ht110116, by Merck Group (1 mentions)
Methyl green, by Vector Laboratories (1 mentions)
Rodent block m, by Biocare Medical (1 mentions)
Rmr622, by Biocare Medical (1 mentions)
Sf994, by Thermo Fisher Scientific (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
11 protocols using xylene substitute
1
Histological Analysis of Tumor Samples
2
Histological Analysis of Tumor Samples
3
Quantifying Lung Metastasis in Mice
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Mice were sacrificed at four weeks (Experiment NO 1 and 2 in vivo) after secondary injection and the lung tissue was dissected. The tissue was fixed in 10% formalin at room temperature overnight and consequentially dehydrated in 70% ethanol. Samples were mounted in paraffin blocks and sectioned at 5 mm of thickness. Slides were baked at 60 ℃ for 15 min, then treated with Xylene Substitute (Citrisolv) (Fisher chemical cat. x3p-1GAL) then rehydrated and stained with Hematoxylin, followed with washes with running water. An incubation with Acid Alcohol (Sigma, cat. A3429) was performed, followed with washes with running water. The samples were then stained with eosin (Thermo Scientific) then dehydrated with ethanol followed by Xylene Substitute (Citrisolv). Samples were mounted with Permount (Fisher Scientific, cat. SP15-500). Slides were submitted for pathologic evaluation. Pathologist was blinded to the sample.
The mouse weight was recorded for analysis and lungs were collected for H&E staining. Pulmonary metastasis was evaluated by visually counting the number of metastatic nodules, maximum diameter per metastatic nodule and cross-section area per metastatic nodule in the entire mouse lung section for each mouse, using Nikon NIS-Elements software (Nikon Corporation Instruments, Tokyo, Japan).
The mouse weight was recorded for analysis and lungs were collected for H&E staining. Pulmonary metastasis was evaluated by visually counting the number of metastatic nodules, maximum diameter per metastatic nodule and cross-section area per metastatic nodule in the entire mouse lung section for each mouse, using Nikon NIS-Elements software (Nikon Corporation Instruments, Tokyo, Japan).
4
Histological Analysis of Bhlhe40-Deficient Tumors
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Tissue samples of liver and lung from Bhlhe40−/− tumor mice were processed using the standard tissue protocol on an Automatic Tissue Processor Donatello (Diapath) and embedded in paraffin. 2-µm paraffin sections were cut. Sections were rehydrated with xylene substitute (Thermo Fisher Scientific) and ethanol, followed by staining with H&E (both Thermo Fisher Scientific) for 1 min each and again dehydrated with ethanol and xylene substitute. After embedding in aqueous mounting medium (Eukitt Neo), slides were analyzed using a Pannoramic FLASH 250 III slide scanner equipped with an Adimec Quartz Q12A180 camera.
5
Tissue Processing and Staining for Bhlhe40 Tumor Analysis
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Tissue samples of liver and lung from Bhlhe40 -/-tumor mice were processed using the standard tissue protocol on Automatic Tissue Processor Donatello (Diapath) and paraffin-embedded. Then 2 µm paraffin sections were cut. Sections were rehydrated with xylene substitute (Thermo Scientific) and ethanol, followed by staining with hematoxylin and eosin (both Thermo Scientific) for 1 min each and again dehydrated with ethanol and xylene substitute. After embedding in aqueous (Eukitt Neo) mounting medium, slides were analyzed using a Pannoramic FLASH 250 III slide scanner equipped with an Adimec Quartz Q12A180 camera.
6
Histological Analysis of Murine Osteosarcoma
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Mice were sacri ced at four weeks (Experiment NO 1 and 2 in vivo) after secondary injection and the lung tissue were dissected. Osteosarcoma specimen of patients were stored at -80℃ after resection. The tissue was xed in 10% formalin at room temperature overnight and consequentially dehydrated in 70% ethanol. Samples were mounted in para n blocks and sectioned at 5 mm of thickness. Slides were baked at 60℃ for 15 minutes, then treated with Xylene Substitute (Citrisolv) (Fisher chemical cat. x3p-1GAL) then rehydrated and stained with Hematoxylin, followed with washes with running water. An incubation with Acid Alcohol (Sigma, cat. A3429) was performed, followed with washes with running water. The samples were then stained with eosin (Thermo Scienti c) then dehydrated with ethanol followed by Xylene Substitute (Citrisolv). Samples were mounted with Permount (Fisher Scienti c, cat. SP15-500). Slides were submitted for pathologic evaluation. Pathologist was blinded to the sample.
The mouse weight was recored for analysis and lungs were collected after H&E staining. Metastasis was measured by visually counting the number of metastatic nodules, maximum diameter per metastatic nodule and cross-section area per metastatic nodule in the entire mouse lung section for each mouse, using Nikon NIS-Elements software (Nikon Corporation Instruments, Tokyo, Japan).
The mouse weight was recored for analysis and lungs were collected after H&E staining. Metastasis was measured by visually counting the number of metastatic nodules, maximum diameter per metastatic nodule and cross-section area per metastatic nodule in the entire mouse lung section for each mouse, using Nikon NIS-Elements software (Nikon Corporation Instruments, Tokyo, Japan).
7
Immunohistochemical Analysis of Ocular Tumor
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Sagittal sections were performed at 4 μm thickness from paraffin blocks of enucleated eyes from in vivo tumor models (n = 6) and human patients (n = 6). Immunohistochemical analysis with human eyes was approved by Institutional Review Board of Seoul National University Hospital (H-1404-032-568) and clinical demographics of 6 patients were summarized in Supplemental Table 1 . We also followed Declaration of Helsinki during the whole procedures with human tissue samples. The sections were incubated at 60°C for 2 hours and processed with deparaffinization and hydrated by sequential immersion in Xylene Substitute (Thermo) and graded ethyl alcohol solutions. Antigen retrieval was performed by the treatment with 0.1 M sodium citrate (pH 6.8, Sigma) at 120°C for 10 minutes. The sections were permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes. Then, we treated the sections with 1X Universal Blocking Reagent (Biogenex) for 10 minutes to minimize nonspecific binding. After incubation with primary antibodies (1:250) overnight, the sections were treated with REAL™ Detection Systems (Dako) and DAB Kit (Life Technologies) as the manufacturer's instructions. Then, the slides were mounted with Permount solution (Thermo) and observed under the light microscope (Nikon).
8
Immunohistochemical Analysis of L1 Expression in Enucleated Eye Tumor Samples
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From paraffin blocks of enucleated eyes from 30 patients, sagittal sections were prepared at 4 μm thickness. The sections were incubated at 60°C for 2 hours and then deparaffinized and hydrated by sequential immersion in Xylene Substitute (Thermo) and graded ethyl alcohol solutions. Antigen retrieval was performed by the treatment with 0.1M sodium citrate (pH 6.8, Sigma) at 120°C for 10 minutes. The sections were permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes. Then, to minimize nonspecific binding, the sections were treated with 1X Universal Blocking Reagent (Biogenex) for 10 minutes. After incubation with the primary antibody against L1 (1:5000; cat. no. ab24345, abcam) overnight, the sections were treated with REAL™ Detection Systems (Dako) and DAB Kit (Life Technologies) according to the manufacturer's instructions. Then, the sections were mounted with Permount solution (Thermo) and observed under the light microscope (Nikon). The percentage of L1-positive cells in tumor sections were evaluated by an experienced pathologist (Y.H. Kim) and confirmed by another independent observer (D.H. Jo). In addition, the number of Flexner-Wintersteiner rosettes in tumor samples were estimated from direct counting throughout the whole tumor sections.
9
Immunostaining of Brown Adipose Tissue
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4-μm-thick paraffin sections were incubated at 4°C for 2 hours and processed with sequential immersion in Xylene Substitute (Thermo) and graded ethyl alcohol solutions. Then, antigen retrieval was performed by immersion of sections in 0.1 M sodium citrate (pH 6.8, Sigma) at 120°C for 10 minutes. After the permeabilization with 0.2% Triton X-100 for 15 minutes, we treated the sections with 1X Universal Blocking Reagent (Biogenex) for 10 minutes to minimize nonspecific binding. For immunofluorescence staining of vessels in BAT, the sections were incubated with Alexa Fluor 594 isolectin GS-IB4 conjugate (4 μg/mL) overnight. Quantification was performed with captured images (4 randomly selected ones per mice) using Image J by the measurement of the proportions of isolectin B4-positive area per image after 8-bit conversion (n = 3~6). For immunohistochemistry of UCP1 in BAT, the sections were incubated with primary antibody to UCP1 (1:100, cat. no.: ab10983; abcam) overnight and treated with REAL Detection Systems (Dako) and DAB Kit (Life Technologies) as the manufacturer’s instructions.
10
Hematoxylin and Eosin Staining of Paraffin-Embedded Sections
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Paraffin-embedded embryonic sections were deparaffinized and hydrated in decreasing concentrations of ethanol in PBS. The slides were stained in Mayer's Hematoxylin Solution (MHS16, Sigma-Aldrich) for 1-4 min at RT, rinsed in warm running tap water and then in 95% ethanol. The slides were then placed in EosinY alcoholic solution (1:200, HT110116, Sigma-Aldrich):glacial acetic acid for 2 s and rinsed. The slides were immersed in 95% ethanol and xylene substitute (Thermo Fisher Scientific) twice for 3 min each and mounted with mounting medium (Life Technologies). Hematoxylin and Eosin images were acquired using a Zeiss AXIO Imager D2 with Zeiss Axio CAMera ICc5. Zen lite software (Zeiss) was used.
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