Cells were fixed with 10% neutralized formalin (Sigma -Aldrich) for 35 min at 4 °C and washed three times with PBS. Cells were then permeabilized with 0.5% Triton X-100 (Sigma) for 30 min and blocked for at least 2 h with a blocking buffer containing 1% BSA (Sigma), 3% donkey serum (Millipore), and 0.2% Triton X-100 in PBS. All samples were incubated with the primary antibodies overnight at 4 °C, followed by a 2 h incubation at RT with secondary antibodies, and phalloidin rhodamine (Cytoskeleton) to stain filamentous actin (F-actin) (1:140). Finally, 30 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies) (1:1000) for nuclei staining was employed. After three PBS washes, samples were kept in Fluoromount-G mounting media (SouthernBiotech) and stored in a humid chamber at 4 °C until imaged.
Neutralized formalin
Manufactured by Merck Group
Sourced in United States
Neutralized formalin is a laboratory reagent used for the fixation and preservation of biological samples. It is a solution of formaldehyde that has been neutralized to a pH-balanced state. The core function of neutralized formalin is to maintain the structural integrity of cells and tissues, enabling their effective preservation for various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Donkey serum, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fluoromount g mounting media, by Southern Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Vibrating microtome, by Campden Instruments (1 mentions)
Alexa fluor 488 goat anti mouse, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 donkey anti goat, by Jackson ImmunoResearch (1 mentions)
Anti cytokeratin 20, by Agilent Technologies (1 mentions)
Donkey anti rabbit alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Fluoromount, by Zeiss (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Application suite microscope software, by Leica (1 mentions)
5 protocols using neutralized formalin
1
Immunofluorescence Staining of Cellular Structures
2
Histological Examination of Tissue Samples
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In cases where visual lesions were found in mice, tissues were fixed with 10% neutralized formalin (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. The tissue samples were subsequently processed in an automatic tissue processor (Tissue-Tek, Sakura, Tokyo, Japan), embedded in paraffin, sectioned, and stained with standard hematoxylin and eosin (H&E; Abcam, Cambridge, UK) for evaluation. Gram-staining using the modified Brown and Brenn method (ScyTek, Logan, UT, USA) and periodic acid–Schiff (PAS; Abcam, Cambridge, UK) staining were performed, and examined histopathologically.
3
Evaluation of Brain Infarct Area
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The chosen animals from each group (n = 4–5 per group) were sacrificed at 7 days after the H-I insult for an evaluation of the brain infarct area. Brains were cut into 2 mm thick coronal sections using a vibrating microtome (Campden Instruments Ltd., Loughborough, UK). The sections were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO, USA) for 8 min to measure infarct volume. The slices were then fixed in 4% paraformaldehyde and subjected to further analysis. A computerized video camera-based image analysis system was used to measure the cross-sectional areas of unstained tissue (open source image processing program ImageJ, http://imagej.net/index.html , designed by Wayne Rasband, NIH, USA).
A histochemical evaluation of brain damage was performed on brains isolated 7 days after H-I. Animals were anaesthetized with halothane and subjected to intracranial perfusion fixation with 4% neutralized formalin (Sigma-Aldrich, St. Louis, MO, USA). The brains were then removed, postfixed for 4 h in the same fixative, and embedded in paraffin. Paraffin blocks containing brain tissue were cut in serial sections at 10 µm thickness using a rotator microtome (Hydrax M 25, Zeiss). The sections were stained according to the Nissl protocol with 0.5% cresyl violet for the histological assessment of neuronal cell damage.
A histochemical evaluation of brain damage was performed on brains isolated 7 days after H-I. Animals were anaesthetized with halothane and subjected to intracranial perfusion fixation with 4% neutralized formalin (Sigma-Aldrich, St. Louis, MO, USA). The brains were then removed, postfixed for 4 h in the same fixative, and embedded in paraffin. Paraffin blocks containing brain tissue were cut in serial sections at 10 µm thickness using a rotator microtome (Hydrax M 25, Zeiss). The sections were stained according to the Nissl protocol with 0.5% cresyl violet for the histological assessment of neuronal cell damage.
4
Immunofluorescence Staining of Cell Cultures
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Cells were fixed with 10% neutralized formalin (Sigma), permeabilized with 0.5% Triton X-100 (Sigma), and blocked for at least 2 h with a blocking buffer containing 1% BSA (Sigma), 3% donkey serum (Millipore), and 0.2% Triton X-100 in PBS. The primary antibodies used were: antifibronectin (1:300, Sigma), anti-Ki67 (1:100, abcam), anticytokeratin 20 (1:100, Dako), anti-myoD monoclonal antibody (MYOD) (2 µg/ml, Sigma) and anti-Myosin Heavy Chain antibody (MYH1E) (1:5, DSHB). All samples were incubated with the primary antibodies overnight at 4°C, followed by a 1 h incubation at RT with secondary antibodies Alexa Fluor® 488 donkey anti-goat, Alexa Fluor® 488 goat anti-mouse, and Alexa Fluor® 647 donkey anti-rabbit (Jackson ImmunoResearch) diluted at 1:500. Alexa Fluor® 568 phalloidin was used to stain filamentous actin (F-actin). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:1000). Finally, samples were mounted with Fluoromount® and observed using an Axio Observer 7 (Carl Zeiss).
5
Histological Analysis of Hair Regrowth
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Dorsal skin from all mice was fixed in 10% neutralized formalin (Sigma) for 18 h. Fixed skins were dehydrated with ethanol and xylene. 4 μm cut paraffin-embedded sections were prepared with rotary microtome. Hematoxylin and eosin (H&E) solution (YD Diagnostics Corp., Yongin, Korea) was applied to the slides to monitor the hair regrowth. The slides of each group were evaluated by randomly selecting 5 photographs per mice. Digital images were obtained using the Leica Application Suite Microscope Software (Leica Microsystems Inc., IL, USA). The used magnification was × 100.
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