To detect injury to the colon and renal tissues, prepared tissues were cut into 2-μm thick sections using a microtome (Thermo Fisher Scientific, Waltham, MA, USA) and stained with hematoxylin and eosin (HE) for histological investigations. To assess the GBM, periodic acid-Schiff (PAS) was used to estimate the glomerular deposition of matrix glycoprotein [51 (link)]. Briefly, 3-μm-thick sections were stained using a PAS staining kit (Merck, Darmstadt, Germany) and counterstained with hematoxylin according to the instructions from the manufacturer. To detect collagen fiber deposition [52 (link)], 3-μm sections were stained with a Masson’s trichrome staining kit (Muto Pure Chemicals, Tokyo, Japan) according to the manufacturer’s instruction. Microscopic images were acquired using a light microscope with a charge-coupled device camera (Olympus, Tokyo, Japan).
Pas staining kit
Manufactured by Merck Group
Sourced in United States, Germany
The PAS staining kit is a laboratory equipment product used to detect the presence of polysaccharides and glycoproteins in tissue samples. It provides a reliable and standardized method for visualizing these biomolecules through a series of staining steps.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager m1 microscope, by Zeiss (2 mentions)
Axiovision software 4, by Zeiss (2 mentions)
He staining kit, by Beyotime (2 mentions)
Evos xl core microscope, by Thermo Fisher Scientific (2 mentions)
Microtome, by Thermo Fisher Scientific (1 mentions)
Charge coupled device camera, by Olympus (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
24 well plate, by Corning (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Ab92551, by Abcam (1 mentions)
Ab15481, by Abcam (1 mentions)
Ab198294, by Abcam (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Dako real detection system, by Agilent Technologies (1 mentions)
Rabbit anti pig cx43, by Cell Signaling Technology (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Nuclear fast red, by Merck Group (1 mentions)
Axioskop 2, by Zeiss (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
46 protocols using pas staining kit
1
Histological Assessment of Tissue Injury and Extracellular Matrix
2
Alcian Blue Staining of SMSC
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Cover slides were coated with poly-d -lysine (0.1 mg/ml) (Merck Millipore, Billerica, Massachusetts, USA) at 37 °C (5 % CO2) for 60 min, then washed and dried overnight. Afterwards, SMSC were grown on the slides in coculture in 24-well-plates (Corning Incorporated, Corning, NY, USA). For alcian blue staining, the cells were fixed and stained using the PAS-staining Kit (Merck Millipore, Billerica, Massachusetts, USA). Briefly, staining with alcian blue was followed by incubation with periodic acid, Schiff reagent, and hematoxylin, whereupon each step was followed by washing. After mounting, histological pictures were analyzed using an Olympus BX51 microscope (Olympus Deutschland GmbH, Hamburg, Germany) with the software module Stream Motion adjusting only brightness and contrast.
3
Conjunctival Epithelial Characterization
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The ex vivo conjunctival cultures were fixed in 4% paraformaldehyde at 4 °C for 20 min, followed by a triple washing step with phosphate buffered saline. Stored mucin was detected using the PAS staining kit (Merck Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. Presence of MUC5AC (goblet cell marker) and MUC1 as well as CK13 (i.e., epithelial cell marker) were investigated by immunocytochemistry. Briefly, fixed cultures were permeabilized with 1% triton X-100 blocking buffer (30 min) and primary antibodies against MUC1 (Abcam (Cambridge, UK); ab15481, 1:200 dilution), MUC5AC (Abcam (Cambridge, UK); ab198294, 1:500 dilution), and CK13 (Abcam (Cambridge, UK); ab92551, 1:500 dilution) were incubated overnight at 4 °C. Cy3-conjungated donkey-anti-rabbit antibody (Jackson ImmunoResearch, Cambridge, UK) was added for 2 h at 4 °C, followed by a nuclear counterstain using 4′,6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature. Samples were mounted in citifluor and imaged on an UltraVIEW VoX dual spinning disk confocal system (PerkinElmer, Billerica, MA, USA).
4
Histological Analysis of Liver Scaffolds
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The liver tissue and implanted scaffolds were fixed in 4% paraformaldehyde. The paraffin blocks were prepared by fixation, washing, dehydration, clearing, paraffin infiltration and embedding. Liver tissue and the retrieved scaffolds were sectioned (4- and 8-μm-thick sections, respectively) and processed for further experiments. Hematoxylin and eosin (H&E) staining was performed, and the necrotic area was evaluated by a single blinded pathologist. For liver function evaluation, periodic acid-Schiff staining (PAS Staining Kit, 1.01646.0001; Merck Millipore) was performed to analyze glycogen storage capacity. PAS staining was quantified by the immunoreactive score (IRS). The intensity of glucose staining was scored as follows: no reaction received a score of 0, a weak color reaction received a score of 1, a moderate-intensity color reaction received a score of 2, and an intense reaction received a score of 3. In addition, the extent of glucose staining (%) was scored as follows: a lack of positive cells denoted a score of 0, <25% positive cells denoted a score of 1, 25% to 50% positive cells denoted a score of 2, 51% to 75% positive cells denoted a score of 3, and >75% positive cells denoted a score of 4. To obtain the final score, the intensity score was multiplied by the extent score.
5
Quantifying Pulmonary PAS-Positive Material
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Cryopreserved lungs were completely cut in sequential slides (4 μm). Systematic uniform random sampling was applied to randomly select slides to quantify PAS-positive material (total of 8–10 slides per lung). Periodic acid-Schiff (PAS) staining was performed, following the provider's protocol, with a PAS staining kit (Merck) and hematoxylin (Sigma-Aldrich) as nuclear counterstaining. Slides were automatically scanned with an AxioScanZ.1 (Carl Zeiss), and random subsampling (10% fraction) at 20× magnification was automatically done with NewCAST system software (Viosiopharm A/S). Subsampled images were quantified by superimposing a test system (point grid) using the stereological tool Stepanizer (Tschanz et al., 2011 (link)), and PAS-positive material was quantified as a fraction of total lung tissue by point counting.
6
Immunohistochemical Analysis of Cardiac Tissue
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For immunohistochemistry formalin-fixed and paraffin embedded left ventricles were used. For C5aR1 staining polyclonal anti-CD88/C5aR1 (Acris Antibodies, Herford, Germany) was used as primary antibody. For Cx43 staining rabbit anti-pig Cx43 (Cell Signaling Technology, Danvers, MA, USA) was used. Nitrotyrosine staining was performed using rabbit anti-nitrotyrosine (Merckmillipore, Darmstadt, Germany) and caspase 3 staining by rabbit anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA). Dako REALTM Detection System (Dako, Glostrup, Denmark) was used as secondary system. Fast hematoxylin and eosin staining-kit (Morphisto Evaluationsforschung & Anwendung GmbH, Frankfurt am Main, Germany) was used to investigate extravasal bleeding in cardiac tissue sections. PAS staining was performed using PAS-staining-kit (Merckmillipore, Darmstadt, Germany). Signal density was measured in five randomly chosen, distinct fields of vision (100x magnification) from each slide using an Axio ImagerM.1 microscope and the Zeiss AxioVision software 4.9 (Zeiss, Jena, Germany). Results are presented as mean density of each group (arbitrary units).
7
Cardiac Receptor Immunofluorescence and PAS Staining
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Paraffin sections of the respective left ventricles were deparaffinized and rehydrated. Antigen unmasking was performed in 10 mmol/L citrate buffer (pH 6) at 100°C and unspecific binding sites were blocked with 5% goat serum. Specific antigen binding was performed by incubation with the respective first antibody (Supplemental Table 2, Supplemental Digital Content 4, https://links.lww.com/SHK/A907). Specific antibody binding was detected with AlexaFluor488-labeled second antibody (Jackson Immunoresearch, West Grove, Pa). Nuclei were counterstained with Hoechst and sections were mounted. Expression of the respective receptors was analyzed by fluorescence microscopy using an Axio ImagerM.1 microscope and the Zeiss AxioVision software 4.9 (Zeiss, Jena, Germany) with 40× magnification (N.A. 0.75). Fluorescence intensities were evaluated using ImageJ software (National Institutes of Health, Bethesda, Md) and results are presented as mean pixel density of each group.
PAS staining was performed using PAS-staining-Kit (Merckmillipore, Darmstadt, Germany). Signal density was measured using an Axio ImagerM.1 microscope and the Zeiss AxioVision software 4.9 (Zeiss, Jena, Germany) with 40× magnification (N.A. 0.75). Results are presented as mean density of each group (arbitrary units).
PAS staining was performed using PAS-staining-Kit (Merckmillipore, Darmstadt, Germany). Signal density was measured using an Axio ImagerM.1 microscope and the Zeiss AxioVision software 4.9 (Zeiss, Jena, Germany) with 40× magnification (N.A. 0.75). Results are presented as mean density of each group (arbitrary units).
8
Histochemical Analysis of Osteochondral Explants
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hPLAP-stained and quantified osteochondral explants were fixed in 70% ethanol for 48 h and decalcified with 0.5 M EDTA, pH 8.0, for 4 weeks at 4°C. Thereafter, the explants were embedded in TissueTek (Sakura Finetek), shock-frozen in liquid N2, and kept at −80°C until sectioning. Eleven-micrometer-thick cryosections were transferred to 3-aminopropyltriethoxy-silane (APES; Sigma)-pretreated slides. For hPLAP staining, sections were fixed with acetone–methanol (30:70 v/v) for 3 min at −20°C. As the decalcification procedure inactivates the hPLAP marker gene, reactivation had to be performed as described earlier.30 (link) Therefore, all alkaline phosphatases were reactivated with 1% MgCl2 in 100 mM Tris-maleate buffer, pH 9.2, overnight in darkness. To inactivate endogenous alkaline phosphatases, sections were subsequently incubated at 65°C for 35 min. The staining was carried out as described in detail before.29 (link) Nuclear fast red (Sigma) was used as counterstain. For Alcian blue-PAS (periodic acid-Schiff) staining, a PAS staining kit (Merck) was used according to manufacturer's instructions after sections were fixed with 4% PFA (paraformaldehyde) for 5 min at RT. Pictures of the stained sections were taken with a microscope (Axioskop 2; Zeiss).
9
Histological Analysis of Tissue Samples
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The tissue samples were sectioned at 7 µm almost parallel to the wall and were stained with Delafield’s hematoxylin and eosin (Fig. 4a–c ). Some sections were stained with PAS staining kit (1.01646.0001; Merck KGaA) and counterstained with hematoxylin to confirm the presence/absence of milk secretion.
10
Immunohistochemical Analysis of Protein Expression
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Specific antigen binding was determined by incubating sections with the respective primary antibodies for Cx43 (Cell Signalling Technology, Cambridge, UK), C3aR (Bioss, Woburn, MA, USA), and IL-1β (Abcam, Cambridge, UK) overnight at 4°C. A biotinylated IgG (Life Technologies, Carlsbad, CA, USA) was used as secondary antibody. Signal amplification was performed by using the VECTASTAIN® ABC Kit (Vector Laboratories, Burlingame, CA, USA), and signal was obtained by developing sections with the VECTOR® NovaRED™ Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA). Cell nuclei were counterstained in haematoxylin. Sections were investigated by bright-field microscopy using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Results are presented as mean pixel density. PAS staining was performed using a PAS-staining kit (Merck Millipore, Darmstadt, Germany). Signal density was measured using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Results are presented as mean density.
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