We used immunofluorescence staining to analyze the changes of FOXO1 and p-FOXO1 in the liver after ischemia-reperfusion. Patient and mouse liver samples were stained with anti-FOXO1 antibody (1:100; Ab52857; Abcam, Cambridge, UK) and anti-p-FOXO1 antibody (1:100; Ab131339/Ab259337; Abcam, Cambridge, UK). Liver tissue was fixed in 4% paraformaldehyde in PBS (Sigma-Aldrich, St Louis, MO, USA), washed, and stored in 30% sucrose in PBS for 24 h. The samples were embedded into Optimum Cutting Temperature gel (Tissue-Tek, Torrance, CA, USA), frozen (CM30505; Leica, Wetzlar, Germany), and cut into 10 µm sections. The slides were incubated with goat serum (Gibco, Grand Island, NY, USA) blocking buffer for 1 h at room temperature and then incubated with primary antibody at 4°C overnight. After washing, the slides were incubated with secondary antibody at room temperature for 1 h. After washing with PBS for 3 times, the slides were incubated with diamidino-phenylindole for 10 m, dried, and mounted. Image-Pro Plus 5.0 was used to analyze FOXO1 and p-FOXO1 signals.
Cm30505
Manufactured by Leica
Sourced in Germany
The CM30505 is a laboratory equipment product designed for precise control and analysis. It features advanced temperature regulation and monitoring capabilities. The core function of this product is to provide a controlled environment for various scientific experiments and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (2 mentions)
Ab52857, by Abcam (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Palm microbeam, by Zeiss (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Eclipse ni u microscope, by Nikon (1 mentions)
Dapi solution, by Beyotime (1 mentions)
Evos fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
Anti c fos antibody, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Abcam (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
7 protocols using cm30505
1
Analyzing FOXO1 and p-FOXO1 in Liver Ischemia-Reperfusion
2
Amygdala Region-Specific Laser Capture
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Three days after the last CJB test, mice were anesthetized using 2.5% isoflurane in oxygen and decapitated. Brains were immediately recovered and frozen on dry ice. Afterward, brains were cut with a cryostat (Leica CM30505) into 16–18 μm thick slices, aiming at the posterior and anterior parts of the basolateral nucleus of the amygdala (−0.58 to −0.94 and −2.06 to −2.3 relative to bregma, respectively), here on referred to as posterior and anterior (portions of the) amygdala. Brain slices were picked up using membrane slides (Carl ZeissTM Membrane Slides by Thermo Fisher Scientific Inc.) previously exposed to UV radiation, and fixated for 15 min in 47°C before staining with eosin red (8 min) and methylene blue (3 min). Cells from the anterior and posterior amygdala were then collected using a laser capture microscope (P.A.L.M. Microlaser Technologie, PALM® MicroBeam by Zeiss), following Sangha et al. (2012 (link)).
3
Quantifying Bone Marrow Fat Deposition
4
Immunohistochemical Analysis of c-Fos and Parvalbumin in Mouse Brain Tissue
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As previously described (Han et al., 2020 (link)), mice were anesthetized and perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS), and tissues were fixed in 4% PFA at 4°C for 12 h. After dehydration by 30% sucrose, brain tissue was cut into 30-μm-thick sections using a freezing microtome (Leica CM30505, Germany). The posterior portion of the BLA-containing sections was permeabilized with 0.3% Triton X-100 and 5% donkey serum in PBS for 2 h and then incubated with rabbit polyclonal anti-c-Fos antibody (1:1,000, Abcam, Cambridge, United Kingdom, ab190289) or rabbit polyclonal anti-PV antibody (1:1,000, Abcam, ab11427) at 4°C overnight. After washing three times with PBS, sections were incubated with Cy3-labeled anti-rabbit secondary antibody (1:1,000, Absin, Shanghai, China, abs20024) for 1.5 h at room temperature and then given three 10-min washes in PBS. Afterward, sections were incubated with DAPI solution (1:5 for 15 min, Beyotime, Shanghai, China, C1005) for nuclear labeling and coverslipped with anti-fade mounting medium. Images were captured using an EVOS fluorescence microscope (Invitrogen, United States). For c-Fos immunostaining, mice subjected to several behavioral tests were perfused 1.5 h after the tests.
5
Spinal Cord Injury Tissue Fixation and Immunolabeling
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After the rats were initially perfused with PBS solution, a subsequent perfusion was conducted using a 4 % formaldehyde solution. A 2 cm long section of spinal cord tissue from the injury epicenter was continued to be fixed in 4 % formaldehyde at 4 °C for 24 h. The tissue was dehydrated in a sequential manner in 10 %, 20 %, and 30 % sucrose solutions, each for one day. Subsequently, the tissue was embedded in optimal cutting temperature (OCT) compound and stored at −20 °C. Afterward, using a frozen microtome (CM30505 Leica), the spinal cord was cut into 6 μm thick cryosections. All cryosections were washed for 5 min in 1 × PBS to remove OCT. Then, the cryosections were blocked with PBS containing 0.25 % Triton X-100 (Cat# T8200; Solarbio) and 5 % BSA (Cat# A8020; Solarbio) at RT for 1 h.
GFAP (1: 500; Cat# ab279289; Abcam) and NeuN (1: 500; Cat# ab177487; Abcam) were selected as primary antibodies with an incubation time of 2 h at RT. After circling the appropriate range on the slide with a PAP pen, the primary antibodies were added and incubated for 2 h at RT. Goat Anti-Mouse IgG (Alexa Fluor® 555) (1: 400; Cat# ab150118; Abcam) and Goat Anti-Rabbit IgG (Alexa Fluor® 488) (1: 400; Cat# ab150081; Abcam) were used as secondary antibodies for incubation. After washing, sections were covered and slipped with a mounting medium containing DAPI (Cat# ab104139; Abcam).
GFAP (1: 500; Cat# ab279289; Abcam) and NeuN (1: 500; Cat# ab177487; Abcam) were selected as primary antibodies with an incubation time of 2 h at RT. After circling the appropriate range on the slide with a PAP pen, the primary antibodies were added and incubated for 2 h at RT. Goat Anti-Mouse IgG (Alexa Fluor® 555) (1: 400; Cat# ab150118; Abcam) and Goat Anti-Rabbit IgG (Alexa Fluor® 488) (1: 400; Cat# ab150081; Abcam) were used as secondary antibodies for incubation. After washing, sections were covered and slipped with a mounting medium containing DAPI (Cat# ab104139; Abcam).
6
Tissue Preparation for Brain Imaging
7
Quantitative Analysis of Coronal Brain Sections
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For quantification analysis, 10× serial 10 µm thick coronal sections were obtained from a single coronal slice. To this end, slices were incubated overnight in 30% sucrose for cryoprotection before embedding in Tissue Tek and blocked at −20 °C. Next, a drop of Tissue Tek (Sakura, Finetek, Umkirch, Germany Cat# 4583) was added into a plastic embedding mold (Sakura, Finetek, Umkirch, Germany Tissue-Tek Cryomold Intermediate 15 mm × 15 mm × 15 mm Cat. No. 4566). The slice was transferred with a spatula and was placed on the drop of Tissue Tek. Then, the plastic embedding mold was filled with Tissue Tek, and the position of the slice was adjusted to be planar using a brush. The mold was then shortly transferred into cooled 2-Methylbutane, and 10 µm frozen sections were performed with a cryostate (Leica CM30505). Three frozen serial sections were collected on microscope slides (Superfrost Plus, Thermo Scientific, Waltham, MA, USA Cat# 10149870), and slides were then stored at −20 °C until IHC was performed.
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