Senescence-associated β-galactosidase (SA-β-gal) staining is the gold standard method for quantifying cellular senescence (50 (link)), and has recently been shown to sensitively measure changes in the spinal cord following injury in the mouse (28 (link)). Lumbar L4/5 spinal cord samples were isolated, postfixed in 4% paraformaldehyde for 2 hours, cryoprotected, and embedded in OCT medium. Frozen spinal cord sections (20 μm thick) were cut using a cryostat (Leica) and mounted onto Superfrost Plus slides. The Senescence β‑Galactosidase Staining Kit (Cell Signaling Technology, catalog 9860) was used to visualize the senescent cells, per the manufacturer’s instructions. Briefly, the lumbar spinal cord tissue sections were washed with 1× PBS (pH 7.4; 2 × 10 minutes) and were incubated with the SA‑β‑gal staining solution (pH 6.0) in the dark at 37°C (dry incubator; no CO2) for 24 hours. After the incubation period, the sections were washed with 1× PBS (3 × 10 minutes), coverslipped, and visualized using a Zeiss AxioImager M2 imaging microscope. Experimenters were blinded to the surgery group and sex of the mice during quantification.
Superfrost plus slides
Manufactured by Leica
Sourced in Germany, United Kingdom
Superfrost Plus slides are microscope slides designed for optimum adhesion and sample retention. They feature a positively charged surface treatment that enhances the attachment of tissue sections and cells.
Automatically generated - may contain errors
Lab products found in correlation
Cryostat, by Leica (15 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Fluoromount g, by Southern Biotech (4 mentions)
Ix 71s8f 3, by Olympus (3 mentions)
Streptavidin ap conjugate, by Roche (2 mentions)
Nbt bcip, by Roche (2 mentions)
Phospho akt ser473 193h12, by Cell Signaling Technology (2 mentions)
Mouse neuronal class β 3 tubulin clone tuj1, by Fortrea (2 mentions)
Cm3050, by Leica (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Microtome, by Leica (2 mentions)
Cryomicrotome, by Leica (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
Axioimager m2 imaging microscope, by Zeiss (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Cxnft, by Thermo Fisher Scientific (1 mentions)
Cd4 cf 594 rm4 5, by BD (1 mentions)
31 protocols using superfrost plus slides
1
Senescence-Associated β-Galactosidase Assay for Spinal Cord
2
In Situ Hybridization of Mouse Eye Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After mice were perfused with ice-cold 4% PFA/PBS, eyes were dissected out and fixed in 4% PFA/PBS at 4°C overnight. The eyes were dehydrated with increasing concentrations of sucrose solution (15–30%) overnight before embedding in OCT on dry ice. Serial cross sections (12 μm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were washed twice for 10 min in DEPC-treated PBS and permeabilized twice in 0.1% Tween/PBS for 10 min. After blocking at 50°C for 1 h with hybridization buffer (50% formamide, 5 × SSC, 100 μg/ml torula yeast RNA, 100 μg/ml wheat germ tRNA, 50 μg/ml heparin and 0.1% Tween in DEPC H2O), the sections were hybridized with 2 μg biotin-labeled antisense probes at 50°C overnight. The sections were washed three times at 55°C for 10 min with hybridization buffer, 0.1% Tween/PBS, and then blocked in PBS blocking buffer containing 0.1% BSA and 0.2% TritonX-100. The hybridized probes were detected by Streptavidin-AP-conjugate (Roche), and revealed by chromogenic substrate NBT/BCIP (Roche, Basel, Switzerland). Mouse CHOP and BiP probe sequences were from Allen Brain Atlas (http://mouse.brain-map.org/ ).
3
Digoxygenin-Labeled RNA Probes for In Situ Hybridization
4
Immunofluorescent Imaging of Spleen Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleen biopsies were submerged in PLP buffer (1% paraformaldehyde, 75mM sodium phosphate monobasic, 75mM disodium phosphate, 50mM L-Lysine and 10mM sodium periodate) overnight at 4°C, washed three times in PBS and then dehydrated in 30% (w/v) sucrose overnight. Fixed tissues were then stored at -80°C in OCT Compound (Tissue-Tek) until use. A Leica cryostat was used to cut fixed and frozen tissues at 10μm thickness and mounted onto SuperFrost Plus slides. Sections were permeabilised with 0.3% Triton X-100, 0.1M glycine, 0.1% cold fish skin gelatin and 1% BSA in PBS for 10 min, blocked with serum-free protein block (Agilent) for 1hr, and then stained with fluorescently-conjugated mAbs against iNOS Alexa Fluor 488 (CXNFT, ThermoFisher), CXCL9 eFluor 660 (MIG-2F5.5, ThermoFisher), CD4 CF-594 (RM4-5, BD Bioscience) and B220 Pacific Blue (RA3-6B2, BioLegend) for 1hr, all at room temperature. Stained tissue sections were mounted with ProLong Gold Antifade Mountant (ThermoFisher) and imaged using a Zeiss LSM780 confocal microscope. Images were analysed using the Fiji software [81 (link)].
5
In Situ Hybridization of AKT Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult mice were perfused with ice-cold 4% PFA/PBS, and the eyeballs were dissected out and fixed in 4% PFA/PBS at 4 °C overnight. The eyeballs were dehydrated with increasing concentrations of sucrose solution (15–30%) overnight before embedding in optimal cutting temperature compound on dry ice. Serial cross sections (12 µm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were pretreated with protease and then subjected to in situ hybridization with RNAscope Multiplex Fluorescent Detection Reagents V2 according to the manufacturer’s instruction (Advanced Cell Diagnostics, Hayward, CA, USA). Briefly, the sections were hybridized with the probe solution, followed by amplification and probe detection using TSA plus fluorescein/cyanine 5 (PerkinElmer, San Jose, CA, USA). The sections were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were captured by Zeiss LSM 880 confocal laser scanning microscope with 63 × /1.40 Oil DIC (Carl Zeiss Microscopy, Thornwood, NY, USA). RNAscope probes Mm-Akt1, Mm-Akt2, and Mm-Akt1 were purchased from ACD and targeted bases 1130–2560, 2618–3665, and 24–1138 of mouse Akt1, Akt2, and Akt3 mRNA (NCBI reference sequence: NM_009652.3, NM_007434.4, and NM_011785.3), respectively.
6
In Situ Hybridization of STAT1/STAT3 in Leptin Receptor Knockout Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For in situ hybridization (ISH), adult STAT1LepRKO, STAT3LepRKO, STAT1STAT3LepRKO and control mice were anesthetized with isoflurane and then euthanized by decapitation. Whole brains were dissected, flash frozen in isopentane, chilled on dry ice, and stored at −80 °C. 16 μm-thick coronal sections were cut on a cryostat (Leica), thaw-mounted to SuperFrost Plus slides, allowed to dry at −20 °C for one hour and then stored at −80 °C. Slides were then processed for ISH using RNAScope technology per the manufacturer's protocol (Advanced Cell Diagnostics) using either the 2.5 HD Assay-Brown (322310) or the multiplex fluorescent assay (320850) and Stat1 (492731) and Cre (312281-C3) probes. Images of the colorimetric assay were obtained with an Olympus BX51 and Olympus DP80 color camera under 40X objective with a 0.63X C-Mount for a total effective magnification of 25.2X. Fluorescent images were obtained with an Olympus BX53 and QImaging Retiga 6000 monochrome camera under 20X objective. Photoshop (Adobe) was used to adjust white balance (for color images) and adjust levels to remove nonspecific background (for each channel of the fluorescent images). All images from an experiment were processed identically.
7
Retinal Tissue Preparation and Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4% PFA fixed eyes were dehydrated with increasing concentrations of sucrose solution (15%–30%) overnight and embedded in OCT on dry ice. Serial cross sections (12 μm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The retina sections were blocked in the staining buffer (10% normal goat serum (NGS) and 0.1% Triton X-100 in PBS) for 30 minutes followed by primary antibody staining. The following antibodies were used: mouse neuronal class β-III tubulin (clone Tuj1, 1:500 dilution; Covance), phospho-AKT-Ser473 (193H12, 1:200 dilution; Cell Signaling). The sections were incubated with primary antibodies overnight at 4°C and washed three times for 15 minutes each with PBS. Secondary antibodies (Cy2, Cy3 conjugated) were then applied (1:200–400; Jackson ImmunoResearch) and incubated for 1 hour at room temperature. Sections were again washed 3 times for 15 minutes each with PBS before a cover slip was attached with Fluoromount-G (Southernbiotech).
8
Histopathological Analysis of Liver and Jejunum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples of the liver (taken from the right lobe of the liver, lobus hepatis dexter) and the intestine (taken from the middle jejunum, 3 cm) were collected and immediately fixed in 4% formaldehyde solution to investigate and evaluate pathomorphological changes. Fragments of tissues were cut at 3.0 μm, then positioned onto Superfrost Plus slides (Leica, UK) with the orientation core placed up on the slide. All tissue blocks were oriented the same way; then, the entire tissue block was cut with the remaining sections dipped in wax and stored at room temperature. The sections were stained with hematoxylin and eosin according to standard procedures. Pictures were taken using an inverted Olympus microscope IX 71S8F-3 (Tokyo, Japan) at the magnification 10–20 x for liver samples and 10x magnification for jejunum.
9
Tissue Fixation and Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were fixed individually in the 10% neutral buffered formaldehyde. Tissues sections were cut at 3.0 μm and placed onto Superfrost Plus slides (Leica, UK) with the orientation core placed up on the slide. All sections were oriented the same way and the entire tissue block was cut with remaining sections dipped in wax and stored at the room temperature. The sections were stained with hematoxylin and eosin following standard procedures. Photographs were taken using an inverted Olympus microscope IX 71 S8F-3 (Tokyo, Japan).
10
Tissue Preparation for Cryo-Sectioning
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!