Adult C. elegans were washed in M9 buffer and decapitated with a syringe needle to expel the intestine. Tissues were fixed overnight at 4°C in 4% formalin (Sigma-Aldrich HT5011) then blocked for 1 hour at room temperature in PBS+ (1x PBS with 0.1% Triton X-100, 1% bovine serum albumin, 1% donkey serum, and 0.02% sodium azide). Rabbit anti-FLAG primary antibody (Thermo Fisher Scientific, Cat. No. 740001; 1:1,000) and mouse anti-HDEL primary antibody (previously used by (32 (link)) to mark the ER in C. elegans; Santa Cruz Biotechnology, Santa Cruz, CA; 1:250) were diluted in PBS+ and tissues were incubated occurred overnight at 4°C. Secondary antibody incubation with Alexa Fluor 555 goat anti-rabbit IgG (Thermo Fisher Cat no. A21428; 1:8,000 dilution) was for 1 hour at room temperature. Tissues were washed three times for 5 minutes each with PBS with 0.1% Triton X-100 in between antibody incubation periods. The final wash contained Hoechst nuclear stain (Thermo Fisher 33258) at 1:1,000 dilution to visualize nuclei and phalloidin stain (Invitrogen Alexa Fluor 488 Phalloidin) at 1:300. Confocal images were taken with a Nikon Ti2 spinning disk confocal with a Yokohama X1 disk and an Orca Flash4.0 sCMOS (Hamamatsu). Images were acquired in Nikon Elements AR 5.0.
Ht5011
Manufactured by Merck Group
Sourced in United States
The HT5011 is a laboratory instrument manufactured by Merck Group. It is designed for the purpose of conducting precise measurements and analyses within a controlled laboratory environment. The core function of the HT5011 is to provide accurate and reliable data collection capabilities for scientific research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer a1, by Zeiss (3 mentions)
Lsm 800, by Zeiss (2 mentions)
Prolong anti fade reagents, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Hoechst nuclear stain, by Thermo Fisher Scientific (1 mentions)
X1 disk, by Hamamatsu Photonics (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Orca flash 4.0 scmos, by Hamamatsu Photonics (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Axio microscope, by Zeiss (1 mentions)
Microtome, by Leica (1 mentions)
Oil red o, by Merck Group (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Paraffin, by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab192890, by Abcam (1 mentions)
Ab109012, by Abcam (1 mentions)
25 protocols using ht5011
1
Immunostaining of Decapitated C. elegans
2
Paraffin Embedding of Formalin-Fixed Grafts
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Grafts harvested on POD7 were fixed in 10% formalin solution (Sigma, HT5011) and then embedded in paraffin. Sections of 4 µm were made for hematoxylin and eosin (H&E) staining.
3
Liver Tissue Fixation and Embedding
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For hematoxylin and eosin (H&E), Masson trichrome, and immunohistological stainings, livers were fixed with 10% neutral buffered formalin (HT 50-1-1, Sigma-Aldrich) for 2 days. Next, livers were trimmed and embedded in paraffin wax according to the following protocol: 70% ethanol (two times for 1 h); 80% ethanol (for 1 h); 95% ethanol (for 1 h); 100% ethanol (three times for 1.5 h), xylene (three times for 1.5 h), and finally paraffin wax (two times, at 58–60 °C for 2 h).
4
Quantifying p65 Nuclear Translocation
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HeLa, HEK293T, TRAF3-KO, U2OS and Jurkat cells were transfected with 1 or 2 μg of each appropriate expression vector; p65 protein nuclear translocation was analyzed following 25 ng/mL TNF-α cell stimulation for 20 min. Cells were seeded on cover glasses and 24 h post-transfection HeLa, HEK293T, TRAF3-KO, and U2OS cells were fixed with 4% paraformaldehyde/PBS, whereas Jurkat cells were fixed with formalin (HT5011, Sigma-Aldrich), for 20 min. Cells were then permeabilized with 0.5% Triton X-100/PBS for 20 min, blocked with a 5% milk/PBS solution and incubated with appropriate primary and conjugated secondary antibodies. The cover glasses were then mounted in DAPI-containing Fluoromount-G (Southern Biotech). For confocal analyses, slides were examined under a LSM800 (Carl Zeiss MicroImaging) confocal microscope equipped with 63× 1.4 plan apochromat oil-immersion objective using the ZEN software. For epifluorescence imaging, slides were examined under an AxioImager.Z1 microscope (Zeiss) using the Methamorph software. To quantify p65 nuclear translocation, the ratios of the nuclear/total p65 mean brightness staining were calculated using ImageJ software. RGB profiles were calculated using the RGB profiler plugin.
5
Perfused Brain Tissue Sectioning
6
Organoid Culture of OLFM4-Modified RWPE1 Cells
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Organoid culture was performed following the protocol published previously by Drost et al.1 (link) Briefly, 4 × 105OLFM4-wild or OLFM4-knockout GFP reporter RWPE1 cells were placed with 40 µl Matrigel in the center of each well in a 24-well plate. Human organoid culture medium was prepared following the protocol and 0.5 ml was added to each well. Organoid growth was traced from day 1 to day 12 by taking pictures of GFP-expressing single cells using a ZEISS AXIO microscope with either a GFP filter or using a light field. Organoid images were processed with Adobe Photoshop software. For immunofluorescence staining, organoids that had been cultured for 12 days were fixed with 10% formalin solution (Sigma-Aldrich, # HT5011) in PBS at room temperature for 1 h, then changed into 70% ethanol overnight. After paraffin embedding, 5-µm sections of organoids were cut, and paraffin section slides used for fluorescent immunohistochemistry.
7
Histological Analysis of Liver Tissues
8
Histological Analysis of Rat Myocardium
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In order to observe rat myocardium and calculate cardiomyocyte cross-sectional area (CSA), rat myocardium (thickness, 0.3 cm) was fixed in 10% formalin at 37°C for 24 h (cat. no. HT5011; Sigma-Aldrich; Merck KGaA), dehydrated by ethanol, blocked by paraffin (cat. no. 1.07150; Sigma-Aldrich; Merck KGaA) and sliced (5-µm thick). Later, the sliced rat myocardium was dewaxed by soaking in xylene (cat. no. X749400-250g; Casmart) twice (5 min each). Rehydration was performed via soaking in gradient ethanol (100, 95, 85 and 75%; 2 min each) and rinsing with water. The cells were stained with 5% hematoxylin (cat. no. H9627-100G; Casmart) at 37°C for 15 min, then soaked in 5% acetic acid (cat. no. A465500; Casmart) to be differentiated and soaked in distilled water for 20 min to develop blue. Then, the cells were stained by 0.5% eosin (cat. no. 170; Casmart) at 37°C for 10 min, then rinsed with distilled water at 37°C for 2 sec. Dehydration was performed with graded ethanol (75%, 1 min; 85%, 1 min; 90%, 1 min; 100%, 2 min; and 100%, 4 min). Cells were soaked in xylene twice (5 min each), then sealed by neutral resin (cat. no. XY-23474-1; Casmart) at 37°C for 2 h for observation using a light microscope (CX43; Olympus Corporation).
9
Quantification of Skeletal Muscle Histology
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Ten micrometer thick cross sections of skeletal muscle lying underneath the skin of compressed regions were prepared in a freezing cryostat at −20°C. Air-dried sections were then fixed with 10% formalin (HT-5011, Sigma-Aldrich, St Louis, MO, USA) at room temperature for 10 min followed by counter-staining in Mayer's haematoxylin solution (MHS-1, Sigma-Aldrich) for 45 min and 1% eosin in CaCl2 (31,8906, Sigma-Aldrich) for 1 min. Numbers of interstitial nuclei and muscle nuclei and area of interstitial space were quantified through the use of Image J software of National Institutes of Health. All the histological data were presented as average results from three random, non-overlapping image fields captured under a 20x objective.
10
Quantifying Autophagy Markers in Tissue
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On Day 7, following the different treatments stated above, samples were fixed in formalin solution (Sigma, HT50-1-1) for 48 to 72 h. After fixation, the explants were embedded in paraffin, and slices of 5 µm of thickness were studied. The slices were stained with primary antibodies against LC3B (ab192890, ABCAM, Cambridge, UK) or SQSTM1 (Abcam, ab109012), followed by a secondary antibody conjugated with Alexa Fluor 488 dye (A11008, Life technologies, Carlsbad, CA, USA). Seventeen to twenty pictures per condition were acquired via fluorescence microscopy, and the intensity of staining in the epidermis was analyzed using proprietary methods in Visilog software. Wilcoxon’s test was used to analyze the results, and a p-value lower than 0.05 indicated a statistically significant difference.
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