Immunostaining was performed as previously described [16 (link)]. Briefly, human thyroid samples were fixed in 4% paraformaldehyde and then embedded in paraffin. After rehydration, paraffin sections underwent antigen retrieval by heating in citrate buffer (not done for HIF-1α staining) and endogenous peroxidase activity was inhibited with 1% H2O2 solution for 20 min. Paraffin sections were blocked with a goat serum (1/50, Sigma-Aldrich®, St. Louis, MO, USA) and Bovine serum albumin (BSA) (Millipore, Merck, Darmstadt, Germany) 5% solution for 30 min at room temperature (RT). Sections were then incubated with primary antibody. Incubation time and dilution varied depending on the primary antibody used, as described in Table 2 . A secondary antibody, Envision (Dako, Carpinteria, CA, USA), coupled to peroxidase and specific for the primary antibody (Table 2 ) was applied to the slides for 1 h at RT. Peroxidase activity was revealed with 3-3′ diaminobenzidine tetrahydrochloride (DAB) (Vector, Burlingame, CA, USA). Sections were then counterstained with Mayer’s hematoxylin and fixed in Entellan mounting medium (Millipore, Merck, Darmstadt, Germany).
Entellan mounting medium
Manufactured by Merck Group
Sourced in Germany, United States
Entellan is a mounting medium used in microscopy and histology. It is a clear, fast-drying medium that helps to mount and preserve specimens on microscope slides. Entellan provides a stable, refractive index-matched environment to protect and maintain the sample integrity during long-term storage and observation.
Automatically generated - may contain errors
Lab products found in correlation
Technovit 7100, by Kulzer (4 mentions)
Bx61 microscope, by Olympus (4 mentions)
Rm2145, by Leica (3 mentions)
Axio imager m1 fluorescence microscope, by Zeiss (2 mentions)
Goat anti mouse igg alexafluor647, by Merck Group (2 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Vector Laboratories (2 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine chromogen, by Agilent Technologies (2 mentions)
Stereo investigator software, by MBF Biosciences (2 mentions)
Bx51wi spinning disk confocal fluorescence microscope, by Olympus (2 mentions)
Vectastain elite abc hrp kit, by Vector Laboratories (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Cryotome, by Leica (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Zen 2, by Zeiss (1 mentions)
42 protocols using entellan mounting medium
1
Immunohistochemical Analysis of Thyroid Samples
2
Cryopreservation and Congo Red Staining
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The brain tissue previously fixed in 4% paraformaldehyde for four days was washed with PBS and then cryoprotected with 50% saccharose for seven days. Thereafter, tissues were immersed in Tissue-Tek® and were frozen with dry ice and were kept at −80 °C until further use. The hemisphere was cut into coronal sections of 40 µm thick using a Leica brand cryotome. The sections were placed in 24-well plates containing 0.1% sodium azide dissolved in PBS, and then were stored at 4 °C. A sample of each group was kept aside to perform Congo red staining. First, sodium azide was removed and the sections were incubated in a 20% Congo red solution in deionized water for 3 min at room temperature with stirring. Thereafter, samples were mounted on gelatinized slides and were dried for 24 h at 37 °C in an oven. Third, each of the slides was dehydrated by means of immersion in ethanol (ethanol 70° for 5 min, ethanol 96° for 5 min, ethanol 100° for 5 min), and subsequently rinsed with xylene for 5 min. Finally, each slide was placed in Entellan mounting medium (Merck Milipore, No. Cat. 107960).
3
Immunolocalization of Schistosome Cholinesterases
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Freshly perfused adult S. mansoni and S. haematobium worms were fixed in 4% paraformaldehyde, embedded in paraffin and sections (7 μm thick) were cut in a cryostat. Following deparaffinization in xylene and rehydration in an ethanol series, antigen retrieval was performed by boiling the slides in 10 mM sodium citrate, pH 6.0, for 40 min followed by a solution of 10 mM Tris, 1 mM EDTA, 0.05% Tween, pH 9.0, for 20 min. All sections were then blocked with 10% heat-inactivated goat serum for 1 h RT. After washing 3 times with PBST, sections were incubated with anti-SmAChE1, anti-SmBChE1, anti-SmAChE2, naïve sera (negative control), S. mansoni or S. haematobium infected mouse sera (positive controls) (1:50 in PBST) overnight at 4°C and then washed again (3 × 5 min each). Finally, the sections were incubated with goat-anti-mouse IgG-alexafluor647 (Sigma) (1:200 in PBST) for 1 h in the dark at RT. After a final washing step, slides were mounted with coverslips in Entellan mounting medium (Millipore). Fluorescence and bright-field microscopy were performed with an AxioImager M1 fluorescence microscope (Zeiss) using 10× and 20× objectives.
4
Anatomical Localization of Sh-TSP in Schistosoma haematobium
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Immunohistochemistry was performed to determine the anatomic sites of Sh-TSP expression in sections of adult worms. Adult worm sections from S. haematobium were de-paraffinized using 2 x 3 min washes each of 100% and 50% xylene and rehydrated in an ethanol series. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM sodium citrate, pH 6) for 40 minutes followed by Tris buffer (10mM Tris, 1 mM EDTA, 0.05% Tween, pH 9.0), for 20 minutes. Subsequently, sections were blocked with 10% goat serum for 1 hour at RT. After washing 3 times with TBS/0.05% Tween-20 (TBST), sections were incubated with anti-Sh-TSP antisera (diluted 1:50 in 1% BSA/TBST) overnight at 4°C and then washed with TBST (3 x 5 min). Sections were finally probed with goat-anti-mouse IgG-Alexa Fluor 647 (Sigma-Aldrich, USA) (diluted 1:200 in 1% BSA/TBST) for 1 h in the dark at RT. After a final washing step with TBST, slides were mounted with Entellan mounting medium (Millipore, Germany) and covered with coverslips. The images were acquired by Nuance software with an AxioImager M1 fluorescence microscope (ZEISS, Germany).
5
Fixation and Sectioning of Plant Inflorescences
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Inflorescences were fixed with FAA [50% (v/v) ethanol, 5% (v/v) acetic acid, 3.7% (v/v) formaldehyde] overnight, dehydrated in a graded series of 50%, 60%, 70%, 85%, 95% and 100% of ethanol for 1 h each, then further incubated overnight in 100% ethanol. The samples were then incubated in 50% ethanol/50% Technovit 7100 base liquid (v/v) for 4 h and then in 25% ethanol/75% Technovit 7100 base liquid (v/v) overnight. The samples were infiltrated in Technovit 7100 infiltration solution (1 g hardener I in 100 ml Technovit 7100 base liquid) with vacuum for 2 h and then incubated for 6 days. All steps above were conducted at room temperature (RT) with gentle agitation. The samples were polymerised with Technovit 7100 polymerisation solution (100 µl Technovit 7100 hardener II in 1.5 ml infiltration solution) at RT for 6 h. Transverse sections of 3 µm were cut using a Leica Microtome HM355S.
For histological analysis, the sections were stained with 0.01% (w/v) acriflavine in H2O for 5 min, mounted in VECTASHIELD (Vector Laboratories) and observed using a TCS SP5 confocal microscope (Leica) with excitation at 488 nm and emission at 492-551 nm.
Alternatively, the sections were stained with 0.05% (w/v) Toluidine Blue in H2O for 1 min, mounted in Entellan mounting medium (Sigma-Aldrich) and observed under a Zeiss Axio Imager M2 microscope.
For histological analysis, the sections were stained with 0.01% (w/v) acriflavine in H2O for 5 min, mounted in VECTASHIELD (Vector Laboratories) and observed using a TCS SP5 confocal microscope (Leica) with excitation at 488 nm and emission at 492-551 nm.
Alternatively, the sections were stained with 0.05% (w/v) Toluidine Blue in H2O for 1 min, mounted in Entellan mounting medium (Sigma-Aldrich) and observed under a Zeiss Axio Imager M2 microscope.
6
Quantitative Analysis of Amyloid Plaques in AD Rats
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Amyloid plaques were visualized in untreated and treated later-stage AD rats using the anti-Aβ mouse monoclonal antibody, McSA1 (Supplementary Table 3 ) specific for aggregates of 39- to 43-amino-acid-long Aβ peptides. Sections were incubated with the primary antibody, McSA1, overnight before incubation with the secondary antibody, biotinylated goat anti-mouse. The sections were finally incubated in the avidin-biotin complex (ABC; Vectastain ABC kit; Vector Laboratories, Newark, CA, USA), mounted on SuperFrost Plus Adhesion Slides, and left to dry overnight on a heating plate. The mounted sections were then de-fatted in xylene and coverslipped using Entellan mounting medium (Sigma-Aldrich).
Glass slides were digitalized using Slide Scanner Axio Scan.Z1 and processed using the software Zen 2.6 (Carl Zeiss Microscopy GmbH, Jena, Germany). A total of 7 brain sections from 1 of the 6 series (intersection distance 240 µm), approximately within the bregma range from –3.14 to –4.52,29 were analyzed for plaque pathology in the hippocampal and cortical areas utilizing Fiji software (Version 1.53j; National Institutes of Health, Bethesda, MD, USA) and a tailored script. For more details, seeSupplementary Materials .
Glass slides were digitalized using Slide Scanner Axio Scan.Z1 and processed using the software Zen 2.6 (Carl Zeiss Microscopy GmbH, Jena, Germany). A total of 7 brain sections from 1 of the 6 series (intersection distance 240 µm), approximately within the bregma range from –3.14 to –4.52,29 were analyzed for plaque pathology in the hippocampal and cortical areas utilizing Fiji software (Version 1.53j; National Institutes of Health, Bethesda, MD, USA) and a tailored script. For more details, see
7
PEDV Spike Protein Immunohistochemistry
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Ileum or colon tissue sections were deparaffinized in xylene and rehydrated with water containing descending concentrations (100%, 95%, and 70%) of ethanol. The slide was immersed in a citric acid (pH 7.4)–sodium citrate buffer solution (pH 8.0) at 121°C for 30 min for antigen retrieval. The tissues were rinsed with running tap water for 5 min and in PBS (Gibco) for 5 min, immersed in a 3% (vol/vol) H2O2–methanol solution for 30 min to block endogenous peroxidase activity, rinsed with running tap water for 5 min, and then blocked with 5% skim milk (Sigma-Aldrich, USA) in PBS for 30 min at room temperature. Mouse anti-PEDV spike protein monoclonal antibody (mAb) 6E5 (stocked in our laboratory) was diluted 1:200 in PBS. After overnight incubation at 4°C, the tissue sections were subjected to three 10-min washes with PBS. Subsequently, the slides were incubated with goat anti-rabbit secondary antibody in the dark for 30 min at room temperature, subjected to three 5-min washes with PBS, visualized rapidly using diaminobenzidine (DAB) (Sigma-Aldrich, USA), and counterstained with hematoxylin. The slides were then dehydrated with ascending concentrations (70%, 95%, and 100%) of ethanol, clarified in xylene, and mounted with Entellan mounting medium (Sigma-Aldrich, USA). Staining was observed using an optical microscope.
8
Histological Analysis of Colon Tissue
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After the animals were sacrificed, adjacent colon tissue to the one used for MPM was preserved in Roti-Histofix (Carl Roth) and embedded in paraffin. Afterward, tissue samples were cooled at -20°C, and 3-µm slices were cut with a microtome (Leica) and collected on glass slides. The slides were then placed in an incubator at 65°C for at least an hour for the paraffin to melt and allow the tissue binding to the slide. The samples were then deparaffinized in Rotihistol (Carl Roth) and rehydrated in 100%, 96%, and 70% ethanol. Further, they were shortly washed in water and the cell nuclei were stained in Harris hematoxylin solution (Carl Roth). Counterstaining of cytoplasm was done by immersing the samples in eosin staining solution (Merck). The cuts were then dehydrated in 70%, 96%, and 100% ethanol. To complete the tissue dehydration, the slides were immersed in Rotihistol and mounted with Entellan mounting medium (Sigma Aldrich). The H&E stainings were then evaluated with the DMi4000B inverse microscope (Leica) at 10× and 20× magnification. Thus, the field of view in 2-dimensional H&E images was larger compared with the 3D stacks of MPM (25×, see the following).
9
Mass Spectrometry Imaging Protocol
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2,5-dihydroxybenzoic acid (2,5-DHB), ammonium
bicarbonate (ABC), ammonium formate (AF), dithiothreitol (DTT), Entellan
mounting medium, ethanol (EtOH), eosin-Y, formic acid (FA), Gill’s
hematoxylin, iodoacetamide (IAM), methyl-tert butyl ether (MTBE),
and xylene were purchased from Sigma-Aldrich (The Netherlands). 2-propanol
(IPA), acetonitrile (ACN), methanol (MeOH), trifluoroacetic acid (TFA),
and water (H2O) were purchased from BioSolve (The Netherlands). The
RapiGest (RG) surfactant was purchased from Waters (USA). Enzyme mix
(trypsin/Lys-C) was purchased from Promega (United States). PEN-membrane
slides and Pierce FlexMix Calibration Solution (A39239) were purchased
from Thermo Fisher (The Netherlands). ITO slides were purchased from
Delta Technologies (USA). IntelliSlides were purchased from Bruker
Daltonik GmbH (Germany). MSI SPLASH mix (330841) was purchased from
Avanti Polar Lipids (USA) (detailed composition is given inTable S1 ). All reagents were of LC-MS grade.
bicarbonate (ABC), ammonium formate (AF), dithiothreitol (DTT), Entellan
mounting medium, ethanol (EtOH), eosin-Y, formic acid (FA), Gill’s
hematoxylin, iodoacetamide (IAM), methyl-tert butyl ether (MTBE),
and xylene were purchased from Sigma-Aldrich (The Netherlands). 2-propanol
(IPA), acetonitrile (ACN), methanol (MeOH), trifluoroacetic acid (TFA),
and water (H2O) were purchased from BioSolve (The Netherlands). The
RapiGest (RG) surfactant was purchased from Waters (USA). Enzyme mix
(trypsin/Lys-C) was purchased from Promega (United States). PEN-membrane
slides and Pierce FlexMix Calibration Solution (A39239) were purchased
from Thermo Fisher (The Netherlands). ITO slides were purchased from
Delta Technologies (USA). IntelliSlides were purchased from Bruker
Daltonik GmbH (Germany). MSI SPLASH mix (330841) was purchased from
Avanti Polar Lipids (USA) (detailed composition is given in
10
Cresyl Violet Staining for Brain Visualization
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In order to visualize brain structures, every 10th microscope slide (containing 4 sections at 250-μm distance from each other), was stained using standard cresyl-violet staining protocol, as previously described (Jakovljević et al., 2022 (link)). Briefly, the sections were rinsed for 3 min in 2 changes of xylene, 95, and 70% ethanol and distilled water. The sections were incubated in cresyl violet dye (Sigma) for 10 min at 60°C, rinsed in distilled water and incubated in a series of ethanols with concentration gradient 70, 95, 100%, 3 min each, and finally cleared in xylene for 5 min, prior to drying and applying Entellan mounting medium (Sigma).
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