Cytochrome c type 3

Manufactured by Merck Group

Cytochrome C (Type III) is a laboratory product offered by Merck Group. It is a protein that plays a crucial role in cellular respiration, specifically in the electron transport chain. The core function of Cytochrome C is to facilitate the transfer of electrons between the various complexes of the electron transport chain, which is a crucial step in the process of ATP production within the mitochondria of eukaryotic cells.

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5 protocols using cytochrome c type 3

Cytochrome Oxidase Staining in Rat Brains

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Two rats were used to verify the possibility of using the technique in this series of experiments. Animals were perfused and tissues postfixed and cut as described above. Brains were freeze sectioned in the sagittal plane. Two consecutive sections, one per stereotaxic plane, were used in two different ways. The first sections (60 μm thick) were incubated at 37°C in the dark for 10–12 h in a solution containing 50 mg DAB, 30 mg cytochrome C (Type III, Sigma), and 4 g sucrose dissolved in 90 mL PB (0.1 mmol/L, pH 7.4; Wong-Riley 1979 (link)). Incubation was arrested when a clear differentiation between cerebral cortex and cc was visible. Sections were rinsed many times in PB, mounted on subbed slides, air-dried, dehydrated in xylene and then coverslipped. The adjacent sections (40 μm thick) were counterstained with neutral red (1% in aqueous solution) and then coverslipped. Selected sections from CC-NADPH-1/11, CC-nNOS-1/6 and CC-Fl-1 and -2 were used for CO staining as described above.

Barbaresi P., Fabri M, & Mensà E. (2014). Characterization of NO-producing neurons in the rat corpus callosum. Brain and Behavior, 4(3), 317-336.

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Polyacrylamide Gel Electrophoresis Protocol

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Acetic acid ca. 100% (Bang & Co.) or Emprove (Merck). Acrylamide (99.9%) and N,N′-methylene-bis-Acrylamide (electrophoresis purity) (Bio-Rad). Agarose (GTG Agarose, Biometra). Ammonium persulfate (electrophoresis purity) (Bio-Rad). β-Mercaptoethanol puriss (Fluka). Boric acid p.a. (Merck). Bromophenol blue (Merck). Coomassie Brilliant Blue R 250 (Serva). Cytochrome-C, type III (Sigma C-2506). DL-dithiothreitol (Sigma D-0632). DNA from Calf Thymus Type I, Na salt, highly polymerized, (Sigma D-1501). Ethidium bromide (Sigma E8751). Ethylenediamine tetraacetic acid disodium salt p.a. (Merck). Gel Drying Films (Promega). Glycine p.a. (Riedel-deHaën 33226). Methanol p.a. or SeccoSolv (Merck). Myoglobin from Horse Heart (Sigma M-9267). Plasmid pCaMVCN (Pharmacia) 4177 bp, linearized with Cla I (NEB). Precision Protein Standards (Bio-Rad 161-0362) with given molar masses of 250, 150, 100, 75, 50, 37, 25, 15, & 10 kDa. 2-propanol, p.a. Emsure or SeccoSolv (Merck). RNA, soluble from Yeast, Type III (Sigma R-7125). Sodium dodecyl sulphate (BDH 44215). TEMED (electrophoresis purity) (Bio-Rad). Tris, Trizma preset pH, base and HCl (Sigma).

, & Ahokas H. (2019). Staining of RNA and DNA on electrophoretic gels and in cytology with juice of Vaccinium myrtillus berries. Heliyon, 5(10), e02666.

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Cytochrome Oxidase Staining Protocol

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CO staining followed procedures described previously (Horton and Hedley-Whyte, 1984 (link); Murphy et al., 1995 (link); Wong-Riley, 1979 (link)) , with some modifications; sections were incubated in a solution containing 100 mg diaminobmuenzidine, 60 mg cytochrome C (type III, Sigma) and 40 mg catalase per 100 ml of 0.1 M phosphate buffer. The reaction usually took 5d at 39°C. The incubation solution was refreshed daily.

Eilam R., Aharoni R., Arnon R, & Malach R. (2016). Astrocyte morphology is confined by cortical functional boundaries in mammals ranging from mice to human. eLife, 5, e15915.

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Cytochrome C Oxidase Staining for Brain Sections

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Whole brains were fixed in 1X phosphate buffered saline (PBS) / 4% formalin at 4°C overnight, cryoprotected by soaking in 1XPBS / 30% sucrose at 4°C for 3 days, embedded in Tissue-Tec O.C.T. reagent and frozen on dry ice, and stored at −80°C. Brains were sectioned into 40 μm sagittal sections at −25°C with a Leica CM3050 S cryostat. Slices were mounted on Superfrost Plus slides (Fisher Scientific). COX staining was performed as described by Wong-Riley [51 (link)]. Briefly: sections were washed with 0.1 M potassium phosphate buffer pH7.4 (KP_i), then incubated at 37°C in the dark with gentle rotational movement in staining solution consisting of 2.6 mM diaminobezidine (Sigma), 20 μM cytochrome c (type III, Sigma), 130 mM sucrose in KP_i. Color development was stopped by washing in 1XPBS. Air dried sections were mounted with DPX (Sigma) and covered. Slides were imaged with a brightfield microscopy (Keyence BZX710) using identical settings for all samples. Control sections, treated with staining solution lacking either DAB or Cytochrome C, did not stain.

Johnson S.C., Kayser E.B., Bornstein R., Stokes J., Bitto A., Park K.Y., Pan A., Sun G., Raftery D., Kaeberlein M., Sedensky M.M, & Morgan P.G. (2020). Regional metabolic signatures in the Ndufs4(KO) mouse brain implicate defective glutamate/α-ketoglutarate metabolism in mitochondrial disease. Molecular genetics and metabolism, 130(2), 118-132.

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Cytochrome Oxidase Histochemistry in Brainstem

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Four rats from each group were deeply anesthetized and perfused transcardially with 150 ml saline, followed by 50 ml ice-cold mixture of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M PB for 1 h. Brainstems were removed and postfixed by immersion in the same fixative for 4 h at 4mersion in the same fixative for μm thicknesses were prepared with a vibratome (VS1000s, Leica). Sections (n = 18–20) including the pre-BötC region were collected from each brainstem. The basic protocol for CO histochemistry was as described previously (Liu et al., 2001 (link)). Briefly, sections were incubated in 0.1 M PB containing 25 mg 3, 3’-diaminobenzidine (DAB, Sigma), 15 mg cytochrome c, type III (Sigma), and 2 g sucrose per 50 ml solution. They were incubated at 37 for 3 h in the dark. All sections from three groups were reacted together to avoid differences due to slight variations, such as temperature, medium composition, or incubation time. After CO histochemistry, sections were washed three times, 5 min for each in cold 0.1 M PB. As CO-negative control experiment, two NOR rats were perfused and sectioned as above, and performed SST and NK1R immunocytochemistry procedures, avoiding CO histochemistry.

Kang J., Lu N., Yang S., Guo B., Zhu Y., Wu S., Huang X., Wong-Riley M.T, & Liu Y.Y. (2023). Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry. Frontiers in Cellular Neuroscience, 17, 1132241.

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