The following antibodies and concentrations were used: guinea pig anti-perilipin 1:1500 (Fitzgerald 20R-PP004); rabbit anti-F4/80 1:500 (Santa Cruz M-300); donkey anti-rabbit Alexa 647 1:200 (Invitrogen); and goat anti-guinea pig Alexa 488 1:200 (Invitrogen). Paraffin sections were dewaxed and hydrated in Xylene and 100%–95%–80%–70%–50% Ethanol and ddH2O. Slides were placed in chambers containing 1× R-Buffer Buffer A pH 6.0 solution and antigen retrieval was done using Antigen Retriever 2100 (Electron Microscopy Sciences) for 2 h. Following PBS wash for 5 min, Fx Signal Enhancer (Invitrogen) was added to the slides for 30 min at room temperature. Slides were then blocked for 30 min in PBS containing 10% normal goat serum at room temperature. Primary antibodies were then diluted in PBS containing 10% normal goat serum and added to paraffin sections overnight at 4 °C. Following overnight incubation, slides were washed in PBS and incubated with secondary antibodies diluted in PBS containing 10% normal goat serum for 2 h at room temperature. Washed slides were mounted with Prolong Anti-Fade mounting medium containing DAPI (Invitrogen) before images were acquired for analysis.
Prolong antifade mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Prolong Antifade mounting medium is a product designed to preserve and protect fluorescent signals in microscopy samples. It is a non-hardening, glycerol-based solution that helps maintain the brightness and stability of fluorescent labels over time.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Dm6000b epifluorescent microscope, by Leica (7 mentions)
Lsm 510 meta, by Zeiss (6 mentions)
Anti α tubulin dm1a, by Merck Group (4 mentions)
Axiovert microscope, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Mab377, by Merck Group (3 mentions)
Axio imager z1 microscope, by Zeiss (3 mentions)
Axioskop 2 plus microscope, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Tween 20, by Merck Group (3 mentions)
Optimal cutting temperature compound, by Sakura Finetek (3 mentions)
Antigen retriever 2100, by Electron Microscopy Sciences (2 mentions)
Fx signal enhancer, by Thermo Fisher Scientific (2 mentions)
Fisher superfrost polarized slides, by Thermo Fisher Scientific (2 mentions)
Hoechst dye, by Thermo Fisher Scientific (2 mentions)
Goat anti guinea pig alexa 488, by Thermo Fisher Scientific (2 mentions)
Zen 2012, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
93 protocols using prolong antifade mounting medium
1
Immunofluorescence Staining of Adipose Tissue
2
Immunofluorescence Staining of HeLa Cells
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HeLa cells were grown in a 6-chamber slide (LabTek) to 80% confluence, washed with 1× PBS, and fixed with 4% paraformaldehyde (Fisher) for 10 min. Cells were treated with TNBS buffer (0.1% Triton X-100, 1% FBS, and 0.1% NaN3 in 1× PBS) for 20 min for permeabilization and blocking and then incubated overnight at 4 °C with one of the following primary antibodies: mouse anti-HDAC3 (Santa Cruz Biotechnology), rabbit anti-histone H1.3 (Abcam), or goat anti-Eg5 (Santa Cruz Biotechnology). Negative controls for all experiments were performed using non-immune IgG from the same species as the primary antibody. Following washes with 1× PBS, cells were incubated with secondary antibody: goat/donkey anti-mouse-FITC (Santa Cruz Biotechnology) or goat or donkey anti-rabbit-Texas Red (Santa Cruz Biotechnology) or donkey anti-goat-FITC or donkey anti-goat-TR for 1 h 15 min at room temperature. After the 1× PBS washes, chromosomes were stained with Hoechst (Invitrogen) for 5 min, and Prolong Antifade mounting medium (Invitrogen) was added before sealing the coverslip. The cells were imaged at ×1000 magnification using a Zeiss Axiovert 200 M optical microscope with confocal attachment, and the digital images were analyzed with ImageJ software.
3
Immunofluorescence Staining of p63 in HPECs and HMECs
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Example 4
HPECs, passage 29, and HMECs, passage 32 were grown on sterile glass cover slips and fixed in 4% (wt/vol) paraformadehyde and labeled with the primary (mouse anti-p63, Santa Cruz, sc-863) and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG) according to the manufacturer's protocol. DNA in the cells was stained for 3 minutes at room temperature with 0.5 μg/ml Hoescht (no. 33342) in PBS and washed three times with PBS. Coverslips were mounted on glass slides using ProLong anti-fade mounting medium (Invitrogen) for 1 hour at room temperature and were stored at 4° C. A Zeiss Axioskop microscope (Carl Zeiss, Inc., Thornwood, N.Y.) equipped with a 63× objective lens and a Hammamutsu charge-coupled-device camera was used to image the cells. Images were processed using Openlab 3.0.7 software.
4
ADCC-Induced Cell Death Assay
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Tumor cells were plated onto glass coverslips previously coated with poly-d -lysine (BD Biosciences, San Jose, CA, USA) and allowed to adhere overnight. Cells were incubated in ADCC conditions (antibody alone, PBMC alone or PBMC+antibody) at a 100:1 E:T ratio for 4 h. Coverslips were washed 2x with media, fixed with 4% paraformaldehyde and permeabilized in a 0.1% Triton X-100 in 0.1% sodium citrate solution. Coverslips were incubated with In Situ cell death enzyme as per the manufacturer's instructions (In Situ Cell Death Detection Kit, Roche, Basel, Switzerland). Coverslips were mounted with Prolong Anti-fade mounting medium with DAPI (Invitrogen), imaged using the Zeiss Axio Imager microscope and analyzed with Metamorph (Molecular Devices, Sunnyvale, CA, USA) and ImageJ (NIH, Bethesda, MD, USA) softwares.
5
IGF-1R Expression in H2O2-Treated Caco2 Cells
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Immunofluorescence was used to detect IGF-1R in caco2 cells. Caco2 cells were seeded on glass coverslips. After 24 h some of them was treated with H2O2 (100 μM, 12 h) and then cultured in NM or pMSCs conditioned medium. Five days later, all the cells (untreated and H2O2-treated-caco2 cells) were fixed for 20 min at room temperature with 1% paraformaldehyde. After blocking in PBS containing 5% BSA, cells were labeled with anti-IGF-1R primary Abs at 4 °C overnight. Cells were washed three times with PBS and subsequently incubated with the indicated fluorophore-conjugated secondary antibodies for 1 h. Next, cells were labeled with DAPI for nuclear staining and coverslips were mounted on glass slides using prolong antifade mounting medium (Invitrogen). Cells were examined using fluorescence microscopy. Images were acquired by sequential excitation at 488 and 360 nm laser lines. Instrument settings were kept constant for each replicate.
6
Azide-Alkyne Cycloaddition for O-Glycan Detection
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Detection of the azide labeled O-glycans was performed by conjugating the azide to a fluorescently labeled alkyne in a copper-catalyzed azide-alkyne cycloaddition reaction24 (link) as previously described18 (link). Fixed paraffin embedded sections were dewaxed, hydrated, and washed in PBS. Tissue sections were incubated with 10 µl of reaction mix from the tetramethylrhodamine (TAMRA) glycoprotein detection kit (Invitrogen) and incubated at 4 °C overnight. After washing in PBS, the samples were blocked in 5% (v/v) fetal bovine serum and stained with anti-MUC2C3 antiserum (against a C-terminal peptide)25 (link) or anti-Muc17S2 (against a peptide in the SEA domain)26 (link). The specific antibodies were detected with an Alexa 488 conjugated anti-rabbit antibody (Invitrogen) and the sections were mounted using ProLong anti-fade mounting medium (Invitrogen). The images were acquired using a LSM 700 Axio Examiner.Z1 laser scanning confocal microscope, with Plan-Apochromat 20x/0.8 and 40x/1.3 Oil DIC objectives (Zeiss). The images were acquired and processed uniformly using the ZEN 2012 software (Zeiss).
7
Visualizing Intracellular NP Distributions
8
Verifying Electrode Placement in Brain
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In addition to stereotaxic coordinates, the electrode position in the brain was verified in at least 2 animals per recording site by painting a fluorescent dye (DiI, Cat no. 42364, Sigma-Aldrich) on the surface of the electrode prior to penetration. After completing the recordings, the brain was removed, placed in 10% buffered formalin for 5–7 days, and immersed in 30% sucrose solution for two days. The brain was cryosectioned (50 μm) in the coronal plane. After blocking in normal horse serum, slices were incubated in a primary mouse antineuronal nuclei (NeuN) monoclonal antibody (1 : 1000, Chemicon, MAB377), washed three times with phosphate buffered saline (PBS), and incubated with a donkey anti-mouse secondary antibody conjugated to Alexa Fluor 488 (1 : 1000; Invitrogen, A21202). Sections were washed with PBS and mounted on Fisher Superfrost polarized slides and coverslipped with Prolong Antifade mounting medium (Invitrogen). Sections were visualized and photographed with a Zeiss Axio Imager Z1 Microscope equipped with a digital camera, and images were processed with Zeiss AxioVision software. Figure 1 presents the electrode penetration locations (pointed by arrows) in the Str-amygdala (a), HC (b), and Cg (c).
9
Immunostaining Neurons for Stau2 and VGLUT1
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For immunostaining28 (link) neurons were washed with warm Hanks′ Balanced Salt Solution (HBSS) and then fixed with warm 4% PFA in HBSS for 10 min. Fixed cells were washed with HBSS and permeabilized with 0.1% Triton X-100 in HBSS for 5 min. The following antibodies were used: (i) polyclonal antibodies, i.e. selfmade rabbit anti-Stau253 (link), guinea pig anti-VGLUT1 (Synaptic Systems, 419005); (ii) secondary antibodies, i.e. donkey anti-rabbit and goat anti-guinea pig Alexa555 or Alexa647 conjugated (Life Technologies, A31572, A31573, A21450). Coverslips were mounted on slides with Prolong antifade mounting medium (Invitrogen).
10
Cryosectioning and Immunostaining of Mouse Bone and Spleen
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