Tissues were first washed with PBS buffer and adventitia was removed carefully, and then they were fixed with 4% paraformaldehyde (PFA) and embedded in 20% sucrose solution before being frozen in TissueTek cutting medium (Sakura Finetek). 8 μm sections were processed for immunofluorescent analysis. The sections were further fixed with 4% PFA for 20 minutes. For immunostaining of attached cells, cells were fixed in 4% PFA for 20 minutes and permeabilized with 0.1% Triton X-100 (in PBS) for 5 minutes and rinsed for 3 times. Nonspecific binding was blocked by 4% BSA in PBS for 1 hour. Tissues/cells were incubated at 4 °C overnight in incubation buffer containing 4% BSA and the primary antibodies including anti-CDH5 (1:100), anti-Ki67 (Abcam, ab15580, 1:200), anti-F4/80 (BioLegend, 157309, 1:100), anti-SM22α (Proteintech, 10493-1-AP, 1:100), anti-ALDH2 (1:100), anti-ALDH3A1(1:100), anti-ALDH6A1 (1:100), and anti-pSmad2/3 (1:100). After being washed in PBS for 3 times, the specimens were incubated with Alexa Fluor 488-conjugated goat anti-rabbit/-mouse IgG (Abcam, ab150077/ab150113) or Alexa Fluor 555-conjugated goat anti-rabbit/-mouse IgG (Abcam, ab150078/ab150114) or Alexa Fluor® 647 goat anti-rabbit igG (Abcam, ab150083) for 1 hour at room temperature. The fluorescent signals were detected by fluorescence microscopy (Leica TCS SP8).
Tissuetek cutting medium
Manufactured by Sakura Finetek
TissueTek cutting medium is a product designed to assist in the preparation and processing of tissue samples for histological analysis. It provides a supportive matrix to aid in the cutting and sectioning of delicate tissue specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ab15580, by BioLegend (1 mentions)
Anti f4 80, by BioLegend (1 mentions)
Ab150077 ab150113, by Abcam (1 mentions)
Ab150078 ab150114, by Abcam (1 mentions)
Tcs sp8, by Leica (1 mentions)
Oil red o, by Solarbio (1 mentions)
Masson trichrome, by Solarbio (1 mentions)
Hematoxylin eosin, by Solarbio (1 mentions)
Profilin1, by Merck Group (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
Dapi anti fade mounting medium, by Vector Laboratories (1 mentions)
Cm1900, by Leica (1 mentions)
3 protocols using tissuetek cutting medium
1
Immunofluorescent Analysis of Tissue Samples
2
Quantifying Atherosclerotic Lesions in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized and fixed by perfusion through the left cardiac ventricle with 4% paraformaldehyde, then the heart, arterial tree, or ligated carotid arteries were dissected carefully. Left ventricular outflow tracts were fixed in 4% paraformaldehyde and dehydrated in 30% sucrose solution before being frozen in the TissueTek cutting medium (Sakura Finetek). Consecutive 10-µm sections were cut for histological analyses. To quantify atherosclerotic lesions, representative sections where 3 aortic valve leaflets were equally observed were stained with hematoxylin-eosin, oil red O, or Masson trichrome according to the manufacturer’s instructions (Solarbio, China). The volume of lesions was calculated by measuring the lesion areas of each section. Area was defined by the internal elastic lamina to the luminal edge of the lesion. Arterial trees were stained with oil red O, and the plaque sizes were quantified by the Image J software (National Institutes of Health). All analyses adhered to the guidelines of AHA Statement for Animal Atherosclerosis Studies.36 (link)
3
Cryopreservation and Immunohistochemistry of Spinal Cord Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were cryopreserved by incubation in 20% sucrose until tissue sank (1–2 days),
frozen in the TissueTek cutting medium (Sakura Finetek, Torrance, CA). The spinal cords
were cut into longitudinal sections 30 µm in thickness with cryostat (Leica CM1900).
Unspecific binding sites were blocked by incubation with (PBS, 5% FBS, 0.5% Triton
X-100) for 2 hours at room temperature. Primary and secondary antibodies were suspended
in (PBS, 1% FBS, 0.1% Triton X-100). Sections were incubated overnight at 4° C with
primary antibodies: profilin1 (1:1000, Sigma), GFAP (1:1000; Novus Biologicals
NB-300-141), and IBA1 (1:1000; Wako 019-19741) and 2 hours at room temperature with
secondary antibodies (i.e., 1:500, Alexa-Flour 488 or 647; Invitrogen). F/G-actin ratio
was assayed by staining tissues with phalloidin (1:200, Sigma) to detect F-actin and
DNaseI conjugates (1:200, ThermoFisher) to detect G-actin. Sections were mounted onto
glass slides with DAPI/anti-fade mounting medium (Vector Laboratories). Images were
taken with Zeiss Confocal Microscope Confocal LSM 510 (Zeiss, Thornwood, NY).
Quantification of fluorescence intensity was analysed by ImageJ.
frozen in the TissueTek cutting medium (Sakura Finetek, Torrance, CA). The spinal cords
were cut into longitudinal sections 30 µm in thickness with cryostat (Leica CM1900).
Unspecific binding sites were blocked by incubation with (PBS, 5% FBS, 0.5% Triton
X-100) for 2 hours at room temperature. Primary and secondary antibodies were suspended
in (PBS, 1% FBS, 0.1% Triton X-100). Sections were incubated overnight at 4° C with
primary antibodies: profilin1 (1:1000, Sigma), GFAP (1:1000; Novus Biologicals
NB-300-141), and IBA1 (1:1000; Wako 019-19741) and 2 hours at room temperature with
secondary antibodies (i.e., 1:500, Alexa-Flour 488 or 647; Invitrogen). F/G-actin ratio
was assayed by staining tissues with phalloidin (1:200, Sigma) to detect F-actin and
DNaseI conjugates (1:200, ThermoFisher) to detect G-actin. Sections were mounted onto
glass slides with DAPI/anti-fade mounting medium (Vector Laboratories). Images were
taken with Zeiss Confocal Microscope Confocal LSM 510 (Zeiss, Thornwood, NY).
Quantification of fluorescence intensity was analysed by ImageJ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!