Immediately after the SD, five mice per group were sacrificed by decapitation, their brains were dissected out on ice, and the hippocampus was extracted and put promptly in cold (4°C) RNAlater solution (Ambion Chemicals, Carlsbad, CA, USA), and immediately stored at −80°C. The remaining animals (n = 5 per group) were used for immunoflourescence and sacrificed with 100 mg/kg pentobarbital followed by the transcardial perfusion. The mice were perfused with 0.1M phosphate buffered saline (PBS) solution at 37°C, followed by 4% paraformaldehyde in 0.1M phosphate buffer, and their brains were post-fixed overnight at 4°C in the same fixative. Later, the brains were cut at 35 µm using a VT1000E vibratome (Leica, Microsystems), starting from the rostral Bregma, −1.34 mm, to the caudal Bregma, −2.92 mm (Paxinos and Franklin, 2013). Tissue storage was done in PBS plus 0.3% sodium azide.
Vt1000e vibratome
Manufactured by Leica
Sourced in Germany
The Leica VT1000E is a vibratome, a type of microtome used for cutting thin sections of biological samples. It utilizes a vibrating blade to produce high-quality tissue sections for microscopy and other analysis.
Automatically generated - may contain errors
Lab products found in correlation
Stereo lumar v12, by Zeiss (2 mentions)
Lumar 43 filter set, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axiocam mrc5 camera, by Zeiss (1 mentions)
Lsm780 confocal laser scanning microscope, by Zeiss (1 mentions)
5 protocols using vt1000e vibratome
1
Hippocampal RNA Extraction and Brain Fixation
2
Transcardial Perfusion and Brain Fixation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57BL/6J mice were anesthetized with isoflurane (5%, 1 L/min) and given a terminal injection of 115 mg/kg sodium pentobarbitone (Lethobarb, Troy Laboratories, Australia) at 3‐ or 7‐days post‐injury, or in a naïve state. Once deeply anesthetized (absence of pedal withdrawal reflex and deep breathing), animals were transcardially perfused using 4% paraformaldehyde (PFA) in 0.01 M PBS to clear the vasculature and fix the tissue. The cranium was removed and post‐fixed in 4% PFA for 12 h, then moved to 0.02% sodium azide (NaN3) in 0.01 M PBS and stored at 4°C prior to sectioning. The brain was removed from the skull in order to perform coronal sectioning at 40 μm using a Leica Microsystems VT1000E vibratome (USA) and the tissue was stored in a 24‐well plate in PBS azide at 4°C preceding the conduction of immunohistochemistry.
3
Histochemical Analysis of Dactyloides glomerata
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Roots and nodules of D. glomerata were harvested and washed in GUS reaction buffer: either ¼ strength SB buffer (12.5 mM PIPES, 1.25 mM MgSO4, 1.25 mM EGTA, pH 6.9) or 100 mM sodium phosphate buffer (pH 6.8) containing 1 mM EDTA, 0.1% (v/v) Triton X-100 with 0.25 mM K3[Fe(CN)6] or without added ferricyanide. Then, the samples were transferred to GUS reaction buffer containing 1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronide), vacuum-infiltrated three times, each time for 5 min and incubated for several hours up to overnight at 37°C in the dark. Afterwards, the samples were placed in ¼ strength SB or phosphate buffer containing 3% (w/v) paraformaldehyde, 0.1% (v/v) Tween 20 and 0.1% (v/v) Triton X-100, vacuum-infiltrated as described above and incubated overnight at 4°C. After fixation the samples were washed several times in reaction buffer. The fixed nodules were embedded in 3% (v/v) agarose and sectioned on a Leica VT1000E vibratome (Leica Biosystems, Wetzlar, Germany). Sections of 50–70 μm thickness were observed under an Axiovert 200M (Zeiss, Jena, Germany) using bright field microscopy; photographs were taken using an Axio HRC Camera (Zeiss).
4
Immunolocalization of Xyloglucan Transferase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cross-sections (70 µm) of haustoria 3 d after FR light induction were prepared using a Leica VT1000E vibratome (Leica Biosystems, Nussloch GmbH, Nussloch, Germany). Free binding sites were blocked for 30min with 5% (w/v) non-fat milk powder in standard phosphate-buffered saline buffer (1× PBS) (blocking buffer). After washing with PBS, 1:20 dilutions of polyclonal anti-XTH rabbit IgGs or IgGs from the pre-immune serum (both provided by Dr E. Labrador and her group at the University of Salamanca, Spain) in blocking buffer were applied to the sections for 2h followed by three 5min washes with PBS. The sections were then incubated for 1h in the dark with Alexa Fluor 555 Goat Anti-Rabbit IgG (Life Technologies, Carlsbad, CA, USA) (1:200 in blocking buffer) and subsequently washed with PBS. Labelled sections were stained with toluidine blue O in order to quench autofluorescence. Micrographs were taken with a SteREO Lumar V12 equipped with an AxioCam MRc5 camera and the Lumar 43 filter set (all from Carl Zeiss, Jena, Germany).
5
Localization of XET Activity in Cuscuta-Infested Plants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Different stages of Cuscuta infection sites on P. zonale were collected based on morphological characteristics [6 (link)]. Cross-sections (100 μm thick) through the sites were prepared using a Leica VT1000E vibratome (Leica Biosystems, Nussloch GmbH, Nussloch, Germany) and provided confirmation regarding the stage of haustorium development. Localization of XET activity using endogenous xyloglucan as donor substrate was carried out as described by Vissenberg et al. [23 (link)] with minor modifications. Cross-sections were incubated in 5 μM XyGO-SR dissolved in 50 mM Na-acetate (pH 5.5), 300 mM NaCl for 1 h in the dark. Sections incubated in buffer without XyGO-SR served as controls. After 10 min washing in ethanol: formic acid: water (15:1:4), the sections were further de-stained overnight in 5% formic acid. Micrographs were taken with a SteREO Lumar V12 equipped with an AxioCam MRc5 camera and the Lumar 43 filter set (all from Carl Zeiss, Jena, Germany). Exposure times were between 300 and 520 ms. The fluorescence emission spectrum from 560 nm to 690 nm was recorded using a LSM780 confocal laser scanning microscope (Carl Zeiss).
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!