Bees were anesthetized by exposure to CO2. The whole gut or desired gut tissue, for example, pylorus or ileum was dissected using a scalpel. Malpighian tubules were removed from the gut tissue and the presence of a scab was documented using a dissection stereomicroscope (Leica) as described in Engel et al., 2015a (link); Emery et al., 2017 (link). The tissues were placed into 2 ml screw-cap tubes containing glass beads (0.75–1 mm diameter, Roth) and 500–1000 µl PBS depending on the experiment. Homogenization of the sample was done by bead beating (FastPrep-24 5g MP Biomedicals) for 40 s at a speed of 7.5 m/s. Serial dilutions (1:10) were performed for each homogenate and plated onto mTYG or BHI agar. Single colonies were counted to determine the total number of bacteria per gut tissue by multiplying with the dilution factor.
Dissection stereomicroscope
Manufactured by Leica
The Leica dissection stereomicroscope is a precision optical instrument designed for detailed examination and analysis of specimens. It provides a three-dimensional, high-resolution view of the subject, allowing for precise observation and manipulation. The stereomicroscope employs a binocular optical system to create a stereoscopic image, enabling the user to perceive depth and visual information in a natural, lifelike manner.
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Lab products found in correlation
5 protocols using dissection stereomicroscope
1
Bacterial Quantification in Bee Gut
2
Evaluating Neuromuscular Junction Integrity in Zebrafish
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To examine NMJ integrity and myelination, knockdowns were performed in zygotes of the Golden (slc24a5b1/+) Danio rerio strain (ZIRC, OR, USA). Zebrafish embryos and larvae were raised and staged according to standard procedures [28] .
Antisense morpholino oligonucleotides (MOs) were purchased from Gene Tools (Pilomath, OR).
We obtained a previously published [33] Sil1 splice-blocking MO directed against the splice acceptor site of exon 2 (5'-GGTGACTGTGTAAACAGAACAAATC-3'). The Gene Tools standard control-MO targeting a human β-hemoglobin gene (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as a negative control for the effects of MO injection. Zygotes were injected with 6ng of either Sil1 MO or control-MO following standard protocols.
Bright field microscopy images of larvae were captured using a Leica dissection stereomicroscope equipped with a Leica digital camera (model DFC 420C). For immunofluorescent staining of whole mount zebrafish, 5-day post fertilization zebrafish embryos were dechorionated using Pronase E (Sigma Aldrich) and euthanized by anesthetic overdose.
Whole mount staining was performed as described previously [29] , utilizing a mouse anti-SV2
Antisense morpholino oligonucleotides (MOs) were purchased from Gene Tools (Pilomath, OR).
We obtained a previously published [33] Sil1 splice-blocking MO directed against the splice acceptor site of exon 2 (5'-GGTGACTGTGTAAACAGAACAAATC-3'). The Gene Tools standard control-MO targeting a human β-hemoglobin gene (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as a negative control for the effects of MO injection. Zygotes were injected with 6ng of either Sil1 MO or control-MO following standard protocols.
Bright field microscopy images of larvae were captured using a Leica dissection stereomicroscope equipped with a Leica digital camera (model DFC 420C). For immunofluorescent staining of whole mount zebrafish, 5-day post fertilization zebrafish embryos were dechorionated using Pronase E (Sigma Aldrich) and euthanized by anesthetic overdose.
Whole mount staining was performed as described previously [29] , utilizing a mouse anti-SV2
3
Zebrafish Touch-Evoked Swimming Assay
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We used the golden strain (slc24a5b1/+) of zebrafish (Zebrafish International Resource Center). Larvae were raised and staged as described.25 (link) Video recordings of embryos were captured using a CMLN-13S2M camera (ClearView Imaging) mounted to a Leica stereomicroscope (Leica). Light microscopy images were taken with a Leica dissection stereomicroscope equipped with a DFC 420C Leica digital camera (Leica). Touch-evoked swimming response was elicited by touching the embryos with a pipette tip. Measurements of the eye diameter and the head diameter (dorsal-ventral axis) were taken and a ratio between the two was calculated. One-tailed Student’s t test was used to assess statistical significance.
4
Dissection and Imaging of Mosquito Salivary Glands
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Adult female, free-mated mosquitoes were fed a virus/blood suspension at 10 days post-emergence via a modified artificial membrane feeder [44 (link)]. Mosquitoes were proffered a viremic blood meal with a final titer of 1.5 × 107 pfu/mL SINVTaV-GFP-TC [24 (link)] and engorged females were incubated for 30 days at insectary conditions. To harvest SG’s, mosquitoes were placed on a glass petri dish submerged under cold 0.1 M Na cacodylate buffer while viewed through a Leica dissection stereomicroscope. Thumb forceps and insect pins were used to remove the SG’s, which were individually transferred into 96 well plates and incubated in 4% paraformaldehyde/0.1 M Na cacodylate buffer overnight at 4 °C. Salivary glands were surveyed on an Olympus FV1000 confocal microscope (Olympus Corporation, Center Valley, PA, USA) with a 4× objective. Positive-stained glands were moved to welled glass microscope slides in 60% glycerol, coverslips applied and reimaged at higher magnifications.
5
Intradermal Evans Blue Dye Tracking
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P12 mice were anesthetized by brief inhalation of isoflurane. The left hind limb footpad was wiped with 70% ethanol and allowed to dry; then, approximately 50 μL of freshly prepared 1% (wt/vol) Evans blue dye dissolved in 1× PBS (pH 7.4) was injected intradermally into the dorsal food pad with the needle (30G) pointed toward the heel. Fifteen minutes after injection, mice were euthanized and the thoracic cavity was opened to observe flow of dye through the thoracic duct and noted for any abnormal reverse flow into the thoracic lymphatic vessels using a Leica dissection stereomicroscope.
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