Plasmids encoding for shRNAs or cDNA were electroporated into the developing brain at embryonic day 16 (E16) and electroporated as described [56 (link)]. In more detail, timed pregnant Sprague Dawley E16 rats were anesthetized with a ketamine xylazine cocktail administered intraperitoneally, and toe pinch was performed to ensure deep anesthesia. To avoid excessive heating loss during the surgical procedure, an external heating source was provided. For pain management buprenorphine and bupivacaine were administered subcutaneously, before the surgery. The abdominal cavity was opened and uterine horns exposed and trans-illuminated for clear identification of the embryonic brain ventricles. For easy visualization of the DNA in the brain ventricular space, a non-toxic dye (Sigma, F7252) was added to the DNA before surgery and electroporated with a sharpened glass needle. After injection, embryos were subjected to five electric impulses (50V, 50ms each, separated by 1s intervals) delivered by an electroporator (Harvard Apparatus ECM 830), to target the DNA to RGPs in the lateral neocortex. The embryos were returned to the abdominal cavity and the wound was closed. Rats were monitored every day post-surgery and buprenorphine was administered every 12h for the first 48h, for post-operative pain control.
F7252
Manufactured by Merck Group
Sourced in United States
F7252 is a laboratory equipment product. It is designed to perform specific functions within a laboratory setting. The core function of F7252 is to facilitate certain laboratory processes, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
6 protocols using f7252
1
In Utero Electroporation of Rat Neocortex
2
GAG Deposition Analysis via Histology
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safranin-O/fast green staining was performed to evaluate the presence of GAG deposition at a histopathological level. Two pellets per donor and condition were fixed overnight in neutral buffered formaldehyde 4% (Boom BV, Meppel, The Netherlands) supplemented with 1% eosin (115935, Merck, Schiphol-Rijk, The Netherlands). Subsequently, the pellets were embedded in alginate and then paraffin. Sections (5 μm) were stained with Mayer’s hematoxylin (3870, Avantor Performance Materials, Center Valley, PA, USA), safranin-O (58884, Sigma-Aldrich) and, as a counterstaining, fast green (F7252, Sigma-Aldrich).
3
Histological Analysis of Mouse Tissues
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Whole heads of mice at indicated time points were freely dissected and collected, and the samples were fixed in 4% paraformaldehyde and demineralized in 0.5 M ethylenediaminetetraacetic acid for 2–3 weeks in the 4°C environment. Then, the samples were embedded in paraffin and cut to 5 μm thickness for regular hematoxylin and eosin (H&E) staining and to 4 μm thickness for safranin O (Sigma-Aldrich, S2255) and fast green (Sigma-Aldrich, F7252) staining. Briefly, for safranin O staining, slides were incubated in safranin O solution for 5–10 min, which was determined by the degree of orange. Then, the slides were washed in differentiation solution and stained with fast green solution for at least 10 min at 37°C. Subsequently, the stained slides were washed with running tap water and cleared in xylene. Finally, the slides were mounted with resinous mounting medium for observation and image acquisition through inverted microscope.
4
Histological Staining of Cartilage
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Nucleus were stained with Weigert’s solution of 5 min. Slides were next rinsed with tap running water for 3 min and then stained into 0.02% Fast Green for 30 sec (F7252, Sigma), followed by 30 sec into 1% acetic acid. To detect proteoglycan within cartilage, slides were stained with safranin’O solution for 45 min (S2255, Sigma).
5
In vivo Electroporation of Developing Rat Brain
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Plasmids encoding for shRNAs or cDNA were injected into the developing brain at E16 and electroporated as described (Baffet et al., 2016 (link)). In more detail, timed pregnant Sprague Dawley E16 rats were anaesthetized with a ketamine xylazine cocktail administered intraperitoneally, and toe pinch was performed to ensure deep anesthesia. To avoid excessive heating loss during the surgical procedure, an external heating source was provided. For pain management, buprenorphine and bupivacaine were administered subcutaneously, before the surgery. Abdominal cavity was opened, and uterine horns were exposed and trans-illuminated for clear identification of the brain ventricles. For easy visualization of the DNA in the brain ventricular space, a nontoxic dye (Sigma, F7252) was added to the DNA before surgery and injected with a sharpened glass needle. After injection, embryos were subjected to five electric impulses (50 V; 50 ms each, separated by 1-s intervals) delivered by an electroporator (Harvard Apparatus ECM 830) to target the DNA to RGPs in the lateral neocortex. The embryos were returned to the abdominal cavity, and the wound was closed. Rats were monitored every day after surgery, and buprenorphine was administered every 12 h for the first 48 h for post-operative pain control.
6
In utero Electroporation of Murine Embryos
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Wild‐type C57BL/6J pregnant mice carrying E13.5 embryos were anesthetized using initially 4% isoflurane (Baxter, HDG9623), followed by 2–3% isoflurane during the in utero electroporation (IUE) procedure. The animals were injected subcutaneously with the analgesic (0.1 ml of metamizol, 200 mg/kg). The peritoneal cavity was surgically opened and the uterus exposed. Using borosilicate microcapillary (Sutter instruments, BF120‐69‐10), the embryos were injected intraventricularly with a solution containing 0.1% Fast green (Sigma‐Aldrich, F7252) in sterile PBS, 2 µg/µl of pSuper plasmid (either 2 µg/µl of pSuper‐shcon or 1 µg/µl of pSuper‐shCcny and 1 µg/µl of pSuper‐shCcnyl1), 0.4 µg/µl of pCAGGS GFP. The electroporations (six 50‐msec pulses of 28V at 1 s intervals) were performed using a 3‐mm diameter electrode (BTX genetronics Inc., 45‐0052INT). After surgery, mice received Metamizol in drinking water (1.33 mg/ml).
Pregnant mice were sacrificed by cervical dislocation at the indicated time points (E15.5‐E17.5), and embryonic brains were dissected, fixed in 4% PFA, overnight at 4°C and processed for cryo‐sectioning.
Pregnant mice were sacrificed by cervical dislocation at the indicated time points (E15.5‐E17.5), and embryonic brains were dissected, fixed in 4% PFA, overnight at 4°C and processed for cryo‐sectioning.
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