In immunohistochemical analyses, mice were fixed with 4% PFA in PBS. The optic nerves were dissected to analyze neurofilament M and βIII-tubulin immunoreactivity. Coronal sections (thickness of 20 µm) were cut on a cryostat (Microm HM550; ZEISS). In the immunocytochemical analysis of the axons of cultured DRG neurons, DRG neurons were fixed with 4% PFA in PBS and permeabilized with 0.2% Triton X-100 in PBS. Incubation with primary antibody was performed at 4°C overnight, followed by secondary antibody at RT for 1 h. Specimens were mounted in Vectashield mounting medium (Vector Laboratories). Immunofluorescence was observed under an inverted microscope (DMI 6000B; Leica Biosystems) and analyzed using LAS AF software (version 3.2; Leica Biosystems). Confocal microscope images were observed under a confocal microscope (FluoView FV1000 IX81; Olympus) and analyzed using FluoView FV10-ASW 3.1 software (Olympus). Images were taken with a constant exposure time between all the conditions of the same experiment and were processed using Photoshop software (Adobe Systems).
Microm hm550
Manufactured by Zeiss
Sourced in Germany
The Microm HM550 is a high-performance cryostat microtome designed for sectioning of a wide range of frozen tissues. It features a motorized vertical specimen advance and an electronic sectioning system for precise and consistent section cutting.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Dmi6000b, by Leica (1 mentions)
Las af software, by Leica (1 mentions)
Fluoview fv1000 ix81, by Olympus (1 mentions)
F4 80, by Merck Group (1 mentions)
Acetyl histone h3, by Merck Group (1 mentions)
Imager z1 microscope, by Zeiss (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
4 protocols using microm hm550
1
Immunohistochemical Analysis of Optic Nerve
2
Aortic Sinus Cryosection Histology
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For histology and immunohistology transversal cryosections of O.C.T. embedded aortic sinus (6 μm) were prepared (Zeiss MICROM HM 550). For detailed information see Suppl. Materials and Methods section.
3
Cryosectioning and Immunostaining of Enucleated Eyeballs
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Each enucleated eye ball was fixed with paraformaldehyde and then placed in a slurry of optimal cutting temperature (OCT) compound in cryomold before freezing in dry ice and storage in a − 80 °C freezer until ready for sectioning using the Microm HM550 (Carl Zeiss Ltd). Five-micrometer cryosections of day 2 post-operated eye tissues stained with hematoxylin and eosin (H&E) staining was visualized as described previously [14 (link)]. A total of 3 eyes for each condition were evaluated. Acetyl-histone H3, CD45 and F4/80 antibodies were obtained from Merck Millipore (Darmstadt, Germany), BD Pharmingen (San Diego, CA, USA) and Abcam Plc (Cambridge, UK), respectively. Isolectin B4-Alexa Fluor 568 conjugate was from Molecular Probes Inc. (Eugene, OR, USA). Labeling by the primary antibodies was detected using secondary antibodies conjugated to Alexa Fluor-594 (red fluorescence) or Alexa Fluor-488 (green fluorescence), both obtained from Invitrogen Corp. (Thermo Fisher Scientific Inc., Waltham, MA, USA). Nuclei were visualized by mounting the cells in DAPI-containing Vectashield mounting medium (Vector Laboratories, CA, USA). Labeled cells were visualized using the Zeiss Imager.Z1 microscope (Carl Zeiss Inc., USA).
4
Histological and Immunohistochemical Analysis of Vascular Grafts
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Cross-sections of VGs (thickness of 10 µm) were obtained using a Microm HM-550 cryomicrotome (Carl Zeiss, Germany) and used for histological and immunohistochemical examination. Samples were stained with hematoxylin and eosin following a classic protocol (H&E; Biovitrum, St. Petersburg, Russia). A detailed protocol of immunohistochemical staining by primary monoclonal and polyclonal antibodies of type IV collagen (clone PHM-12 + CIV22), α-SMA (clone 1A4/asm-1), and factor VIII (rabbit polyclonal) conjugated with horseradish peroxidase was outlined in manuscript [26 (link)].
The thickness of neointima and calcified area were assessed quantitatively as previously described [27 (link)]. Calcified regions were determined as dark purple regions on H&E sections, as described in manuscript [28 (link)].
The thickness of neointima and calcified area were assessed quantitatively as previously described [27 (link)]. Calcified regions were determined as dark purple regions on H&E sections, as described in manuscript [28 (link)].
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