VCaP cells were fixed in 4% paraformaldehyde and centrifuged onto silanized slides (Sigma, St.Louis, MO) with a cytospin centrifuge. Cells were immunostained with anti-ERG (9FY) and anti-NKX3.1 (Santa Cruz) followed by goat anti-mouse Alexa-488 and anti-goat Alexa-594 secondary antibodies (Invitrogen, Carlsbad, CA). Images were captured by using a 40X/0.65 N-Plan objective on a Leica DMLB upright microscope with a QImaging Retiga-EX CCD camera (Burnaby, BC, Canada) controlled by OpenLab software (Improvision, Lexington, MA). Images were converted into color and merged by using Adobe Photoshop.
Silanized slides
Manufactured by Merck Group
Sourced in United States
Silanized slides are specialized glass slides coated with a silane compound. The silane coating enhances the adhesion of biological samples, such as cells or tissues, to the slide surface, facilitating their analysis and study.
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Lab products found in correlation
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Openlab software, by PerkinElmer (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Prolong gold mounting media, by Thermo Fisher Scientific (1 mentions)
Ab 151757, by Abcam (1 mentions)
Dylight 549 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Genpoint kit, by Agilent Technologies (1 mentions)
Nanodrop nd 1000 uv vis, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rnaseh, by Illumina (1 mentions)
Pfu polymerase, by Thermo Fisher Scientific (1 mentions)
Ampliscribe t7 transcription kit, by Illumina (1 mentions)
N methylisatoic anhydride nmia, by Thermo Fisher Scientific (1 mentions)
Dh5α competent cells, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Ecori, by Promega (1 mentions)
Ms 222, by Merck Group (1 mentions)
Paraplast plus, by Leica (1 mentions)
10 protocols using silanized slides
1
Immunostaining of VCaP Cells
2
TUNEL Labeling of Apoptosis in Brain and Heart
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The Terminal deoxynucleotidyl transferase-mediated (dUTP) nick end Labeling kit, also called the Tunel labeling index, was used to analyze cell injury and apoptosis. Tissue section 4–5 µm thick were placed on silanized slides (Sigma Chemical Co.; St. Louis, Missouri, USA) in a suitable holder.
Using a light microscope (BLUE1600BAL-BAT-Biofocus), the slides were manually counted at 100× magnification. Twenty-five brain fields and 50 heart fields were analyzed. For the heart, 25 were from the right ventricle, and 25 were from the left. Means for each slide and the median for each group were then calculated. The slides were blindly read for each group.
Using a light microscope (BLUE1600BAL-BAT-Biofocus), the slides were manually counted at 100× magnification. Twenty-five brain fields and 50 heart fields were analyzed. For the heart, 25 were from the right ventricle, and 25 were from the left. Means for each slide and the median for each group were then calculated. The slides were blindly read for each group.
3
In Situ Apoptosis Detection in Liver
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For the in situ detection of apoptosis in a single cell, the final identification deoxynucleotidyl transferase (TdT) test was used (TUNEL; Boehringer Mannheim, Germany) [80 (link),81 (link)]. According to the standard established by the Laboratory of Histomorphometry and Lung Genomics at the University of São Paulo Medical School, 3–4 µm thick sections of liver tissue were made and placed on silanized slides (Sigma Chemical Co.; St. Louis, MO, USA) on a suitable support, as previously described by Souza et al. [82 (link)].
4
Multiplexed Immunofluorescence Imaging of Tissue
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For immunofluorescence, 3 μm thick tissue sections adhered to silanized slides (Sigma-Aldrich, St. Louis MO, USA) were used. After heat-induced antigen retrieval in citrate buffer, samples were permeabilized and blocked in a single step incubating them for 30 minutes in PBS with 2% normal goat/porcine serum and 0.5% Triton X-100 (Research Organic, Cleveland, OH, USA) at room temperature (RT). Afterwards samples were washed and incubated at 4ºC overnight with primary antibodies: anti-TMPRSS4 [Santa Cruz Biotechnology/ sc-376415 (1:100)], anti-Tryptase [Abcam/ ab-151757 (1:250)] and anti-Chymase [Abcam/ ab-111239 (1:300)]. The next day samples were washed and incubated for 1 hour at RT with fluorescent secondary antibodies (Alexa Fluor[AF]-488 conjugated donkey anti-Rabbit IgG, Dylight-549 conjugated donkey anti-mouse IgG and AF-647 conjugated donkey anti-goat IgG; (Jackson Immunoresearch, West Grove, PA). Nuclei were stained with NucBlue (Life Technologies, Carlsbad, CA). Samples were mounted with ProLong Gold mounting media (Life Technologies). All experiments included controls with/without primary antibody or with/without secondary antibody. Imaging was performed with an FV-1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) in sequential scanning mode to image each fluorochrome separately.
5
HPV Detection in Paraffinized Tissues
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The paraffinized tissue samples were submitted to the DNA extraction technique (QIAamp DNA Mini Kit, QIAGEN Ltd., Crawley, UK), DNA purity, and quantification test (NanoDrop ND-1000 UV-Vis) and PCR for gene control of human β-globin (PC03 and PC04, Life Technologies). A cancer sample was considered HPV-positive after the nPCR method had been performed in triplicate, with the use of the following primers: GP5+ and GP6+ (Life Technologies).
Histological cuts of 5 μm were placed on silanized slides (SIGMA) and submitted to the in situ hybridization reaction (ISH), using specific biotinylated probes for HPV 16 and 18 (DAKO). The ISH reactions were accompanied by a positive control (Kit GenPoint-DAKO), negative control (Kit GenPoint-DAKO, subtracting the probe), and reaction control, using a normal oral mucosa biopsy.
Histological cuts of 5 μm were placed on silanized slides (SIGMA) and submitted to the in situ hybridization reaction (ISH), using specific biotinylated probes for HPV 16 and 18 (DAKO). The ISH reactions were accompanied by a positive control (Kit GenPoint-DAKO), negative control (Kit GenPoint-DAKO, subtracting the probe), and reaction control, using a normal oral mucosa biopsy.
6
Evaluating Cerebral and Cardiac Injury via TUNEL
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TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling) was applied to evaluate cerebral and cardiac injury. Tissue sections of 4 µm were placed on silanized slides (Sigma Chemical Co, St Louis, Missouri, USA) and subjected to deparaffinization using baths of xylene, alcohol, distilled water, and Phosphate-Buffered Saline (PBS).
The recovery of antigenic sites was obtained by proteinase K and peroxidase blockage by 0.3% H2O2 in methanol. The slices were washed with distilled water and PBS before incubation with 50 µL of TUNEL mixture in a humid chamber for 1 hour and a peroxidase converter for 30 minutes. The slices were washed with PBS and incubated with Diaminobenzidine (DAB) for 10 minutes at room temperature, followed by counterstaining with methyl green. A blinded pathologist evaluated 25 fields from brain tissue, left ventricle, and right ventricle using 100 × magnification.
The recovery of antigenic sites was obtained by proteinase K and peroxidase blockage by 0.3% H2O2 in methanol. The slices were washed with distilled water and PBS before incubation with 50 µL of TUNEL mixture in a humid chamber for 1 hour and a peroxidase converter for 30 minutes. The slices were washed with PBS and incubated with Diaminobenzidine (DAB) for 10 minutes at room temperature, followed by counterstaining with methyl green. A blinded pathologist evaluated 25 fields from brain tissue, left ventricle, and right ventricle using 100 × magnification.
7
Oligonucleotide Synthesis and Modification
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Standard phosphoramidites for oligonucleotide synthesis (DNA, RNA, 2'-O-methyl RNA), 6-FAM phosphoramidite and C6-aminolinker were purchased from Glen Research. Phosphoramidites of LNA, LNA 2,6-diaminopurine riboside and 2'-O-methyl-2,6-diaminopurine riboside were synthesized according to published procedures [43 (link)]. Dimethyl sulfate (DMS) was from Aldrich and N-methylisatoic anhydride (NMIA) was from Molecular Probes. Reverse transcriptase SuperScript III was from Invitrogen. AmpliScribe T7 Transcription Kit and RNase H were from Epicenter. Pfu polymerase and dNTP were from Fermentas. Roche was provider of ddNTP. Restriction enzymes: EcoRI and PstI were from Promega. DH5α competent cells, agarose and HybriSlip hybridization cover were bought from Invitrogen. T4 polynucleotide kinase was product of EURx. Silanized slides were purchased from Sigma.
8
Histopathological Analysis of Placental Tissue
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Histological sections of 4µm thickness were taken from biopsies of paraffin-embedded placentas on silanized slides (Sigma Chemical Co., St. Louis, MO, USA). The histological sections were deparaffinized in xylene baths and subsequently hydrated in decreasing concentrations of ethanol. The slides were stained with hematoxylin–eosin and analyzed under a microscope. The histopathological parameters comparing the placental tissue slides of the mothers ZIKV-infected with or without obesity groups were evaluated by the pathologist.
9
Histological Analysis of Zebrafish Brains
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Zebrafish, previously anesthetized with 0.1% 3-aminobenzoic acid ethyl ester (MS-222, Sigma, St. Louis MO, USA), were sacrificed by decapitation. Brains were exposed after dorsal cranium removal. Heads were fixed by immersion in a modified Bouin's fixative solution (see Lazzari et al., 2019) for 24 h at room temperature. After multiple extended 0.1 M phosphate buffer washes, specimens were decalcified in 0.25 M buffered EDTA, pH 7.4, for 9 days at room temperature, dehydrated, and then embedded in Paraplast Plus (Leica Biosystems, Richmond, IL, USA; melting point 55-57°C). Five μm-thick serial frontal sections were collected on silanized slides (Sigma). Some sections were colored with hematoxylin and eosin. Immunohistochemical detection was carried out on adjacent slides.
10
Cathepsin D Immunohistochemical Staining
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Immunohistochemical staining was performed using labeled streptavidin-biotin technique. About 5 µm sections were made from formalin-fixed, paraffin-embedded tissue blocks and taken into silanized slides (SIGMA, USA). The sections were dewaxed in xylene and rehydrated in graded alcohols. Antigen retrieval was done by the pressure cooker method in a 10 mM citrate buffer (pH 6.0) for 2 minutes. Endogenous peroxidase activity was blocked by covering the tissue sections with 3% hydrogen peroxidase for 15 minutes. Then, the sections were incubated for 8 hours in a humidifying chamber with prediluted polyclonal antihuman cathepsin D antibody at 4°C.
The sections were incubated with biotinylated link (secondary) antibody for 15 minutes. This was followed by incubation with streptavidin-peroxidase for 15 minutes. The antigen-antibody reactions were visualized with the 3,3′-diaminobenzidine chromogen. Tris-buffered saline was used in place of primary antibody in negative control tissue sections. The sections were washed and then lightly counterstained with Harris's hematoxylin, dehydrated and mounted with DPX (synthetic resin made up of a mixture of distyrene (polystyrene), plasticizer (tricresyl phosphate), and xylene).
The sections were incubated with biotinylated link (secondary) antibody for 15 minutes. This was followed by incubation with streptavidin-peroxidase for 15 minutes. The antigen-antibody reactions were visualized with the 3,3′-diaminobenzidine chromogen. Tris-buffered saline was used in place of primary antibody in negative control tissue sections. The sections were washed and then lightly counterstained with Harris's hematoxylin, dehydrated and mounted with DPX (synthetic resin made up of a mixture of distyrene (polystyrene), plasticizer (tricresyl phosphate), and xylene).
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