pellet

tube

1% RNase

TE

Add 100 μl of TE with 1% RNase to the tube containing the pellet. To resuspend, leave at 4 °Covernight, flick to resuspend and spin at 16,000 x g for 3 min before use.

pipette tip

tube

pellet

70% EtOH

Add 100 μl of 70% EtOH, vortex briefly. Spin at 16,000 x g for 5 min. Invert to dry until no drops of ethanol are visible. If the pellet slides, push it back to the bottom of the tube with a pipette tip. It is fine to leave the pellets overnight.

isopropanol

tube

Pipet 450 μl of supernatant (doing your best to avoid any loose tissue) (Figure 3) to a new tube and add 350 μl of isopropanol. Mix by inverting.

extraction buffer

tube

leaf tip tissue

Collect ~2.0 cm of leaf tip tissue into each tube and add 500 μl of extraction buffer. Less is more! The tissue will grind better if the leaf is no longer than the length of the tube (Figure 1). The maximum number of samples for one run is 48 samples.

leaf tissue

small spoon

tube

3.2 mm chrome beads

Load 3.2 mm chrome beads to each tube using the small spoon provided with the beads or yourmethod of choice. Use 4 beads/tube for leaf tissue of grass seedlings less than 4 weeks after germination and 5 beads for tissue of mature leaves of any age as long as they are not senescent. Yo u may collect tissue in advance and store it at -20 °C or -80 °C for at least a year prior to prepping.

DNA samples

tube

nuclease free water.

TE buffer

ellet

Resuspension: Dissolve the pellet in 20-50 μl of TE buffer or nuclease free water. Vortex to dissolve if needed, and centrifuge briefly to collect any droplets to the bottom of the tube. Store DNA samples at -20 °C or -80 °C until used.Note: If the DNA pellet is not colorless or white post 70% ethanol wash, add 20-50 μl of TE buffer or nuclease-free water to resuspend the DNA without disturbing the pellet. Gently pipet the liquid a few times in the tube and collect the supernatant as DNA for downstream processes. Keep the pellet until DNA is quantified. Use the DNA for PCR-based detection, or store in freezer until used.

70% ethanol

paper towel

tubes

Wash the pellet: Pour out the supernatant, and remove remaining supernatant by tapping the tubes upside down on a paper towel. Add 200 μl of 70% ethanol, and centrifuge at 21,000 x gfor 5 min.Note: Consider local regulations for correct handling of isopropanol waste.

isopropanol

tube

Precipitate: Transfer ca. 0.7 volume of supernatant into a new tube, add 0.85 volume of isopropanol (room temperature), and mix by inversion for 20 s. Centrifuge at 21,000 x g for 10 min.Note: Minimize transferring any debris while pipetting the supernatant.

Lysis and debris elimination: Incubate the samples in extraction buffer at 65 °C for 15 min. Vortex once during incubation. Centrifuge at 6,000 x g for 10 min.

extraction buffer

samples

liquid nitrogen

pestle

mortar

sample

tube

gDNA sample

TE

ultrapure water

Add 30 μl ultrapure water or TE to the tube and let it stand for 10 min to dissolve the gDNA sample.Note: Dissolve the sample in ultrapure water for subsequent DNA sequencing. For long long-term storage, dissolvethe sample in 30 μl TE.

tube

isopropyl alcohol

Add to the aqueous phase an equal volume of isopropyl alcohol. Mix by gently inverting the tubeand let it stand at -20 °C for at least 1 h. Centrifuge the sample at 12,000 x g for 15 min

tube

pestle

mortar

liquid nitrogen

pinnae

Harvest 2-3 freshly picked pinnae (total wet mass: 10-20 mg) and grind in liquid nitrogen to fine powder using the thoroughly cooled mortar and pestle. And then transfer the powder into a tube.Note: All equipment should be fully cooled in liquid nitrogen during this procedure. Be careful not to let the liquid nitrogen dry while grinding, and the grinded tissue must remain frozen prior to extraction with CTAB solution. Accidental thawing may result in DNA degradation.

70% ethanol

DNA

glass pipette

Fashion a shepherd's crook from a glass pipette over a flame, fish out the clump of DNA in one piece and gently swirl it in a separate conical filled with 70% ethanol for 1 minute.

NEB Proteinase K

beta-mercaptoethanol (BME)

CTAB buffer

Add 15 ul of CTAB buffer, plus 30 ul of beta-mercaptoethanol (BME), plus 200ul NEB Proteinase K (800 U / ml)

pestle

mortar

liquid nitrogen

young tissue

Grind 3.5 g of young tissue in liquid nitrogen with a mortar and pestle

1xTE

DNA pellet

The DNA pellet does not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.

70% ethanol

pellet

Discard supernatant and wash the pellet by adding 1.8 ml 70% ethanol, vortex thoroughly.

2-propanol

2 ml microcentrifuge tube

Transfer the entire clarified upper aqueous layer to a new 2 ml microcentrifuge tube which contains an equal or half volume of 2-propanol and vortex thoroughly.

lysis solution

MM300 Mixer Mill

glass ball

tissue sample

Eppendorf Safe-Lock microcentrifuge tube

Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball (6 mm) freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. Alternatively, grind the sample in lysis solution.

1,5x CTAB

2 ml tube

In a 2 ml tube, add 800 uL of 1,5x CTAB and 1 ul of Beta-mercaptoethanol to the ground leaf material

Beta-mercaptoethanol

ground leaf material

Dissolve pallet in 1x TE (10 mM Tris, pH8, 1 mM EDTA)

1x TE

liquid Nitrogen

frozen plant material

pallet

Grind frozen plant material (100mg) in liquid Nitrogen

Incubate 1-5 million cells in the labeling medium for 10 min at 37 ˚C. For adhesive cells, apply the labeling medium to the cell culture directly in dishes and incubate cells for 10 min at 37 ˚C. For suspension cells, suspend cells in the labeling medium and incubate them for 10 min at 37 ˚C.

Prepare 500 mM N3-kethoxal stock solution using DMSO. Then prepare the labeling medium by diluting the N3-kethoxal solution into pre-warmed (37 ˚C) cell culture medium to a final concentration of 5 mM. It is critical to pre-warm the medium to facilitate N3-kethoxal dissolution.

Eppendorfs

ethylene glycol

petri dish

1X PBS

sucrose

cryoprotection solution

tubes

metallic cryotome holder

sucrose solution

brain

homogenized tissue

brain

brain

mouse

blood

mouse

mice

When mice have reached their endpoint, euthanize them with a lethal injection of pentobarbital (i.p. 10 mg/kg).

pentobarbital (i.p. 10 mg/kg)

RNAzap (ThermoFisher, CAT No. AM9780)

Pierce BCA Protein Assay Kit (ThermoFisher CAT No. 23225)

RNA

protein

cerebral microvessels

4ºC centrifuge

MiliQ water

Eppendorf

pipette

extraction buffer

Dounce homogenizer

1X PBS

RIPA buffer

1 mM AESBF

50 mL conical tube

extraction buffer

lysis buffer

Trizol

tube

RNAase

bead mill

Dounce homogenizer

liquid nitrogen

30% sucrose

4% PFA

tube

curved forceps

razor blade

1X PBS

ice bath

petri dish

scissors

forceps

4% PFA

1X PBS

perfusion pump

21G needle

rectangular bucket

Trizol LS

EDTA

vacutainer blood collection tube

3 mL syringe

22-25G needle

 Take one of the tubes stored at 4ºC and place the brain slices on a petri dish with 1X PBS. Mount the slices on a glass slide with a brush. A total of 3-6 slices can be mounted on a glass slide, depending on the size of the slices. Ensure that the slices are completely straight and not folded. Make sure to leave enough spaces in the edges for the liquid blocker marker. Allow to thoroughly dry.After slices have dried, draw a rectangle with liquid blocking marker to prevent leakage of solutions. Wash slices 6 times with PBS for 5 minutes. Prepare a blocking solution by adding 250 µL of goat serum, 50 µL of 10% Triton X and complete to a 5 mL volume using 1X PBS. Block slides for 30 min with the blocking solution. Aspirate the blocking solution and apply primary antibody (1:500 dilution, or the appropriate dilution for the antibody, in blocking solution). Let incubate overnight at 4ºC (or as appropriate for the antibody) with a wet paper towel to avoid desiccation.The following day aspirate the primary antibody. Wash slides three times with for 5 min with 1X PBS.CRITICAL STEP: once the slices are thoroughly adhered after the first drying step, do not allow slides to completely dry again because this can compromise the integrity of the tissue and the quality of the staining. Make sure to cover slides completely with the solution.Apply secondary antibody (1:500 dilution, or as appropriate, in blocking solution) and incubate for 2 hours at room temperature (or as appropriate) in a light shielded area with a wet paper towel to avoid desiccation.Aspirate the secondary antibody and wash three times with 1X PBS. This would be an appropriate time to apply DAPI or other water-soluble counterstains. For example, if this was an Alzheimer’s disease mouse model and labeling of amyloid plaques was desired, apply Methoxy-X04 diluted 1:250 in 1X PBS and incubate at room temperature for 15 minutes. Wash 2 times with 80% ethanol. Then, allow slides to thoroughly dry before mounting. Use a mounting medium of preference (we recommend Prolong Gold). Our images were acquired with a Zeiss LSM 710 Confocal, using fluorescently-labeled secondary antibodies (Figure 4).

The half brain stored in sucrose will be used for immunostaining. Extract the brain from the sucrose solution and add to a petri dish. Place the brain in a coronal or sagittal position inside the metallic cryotome holder with OCT compound as an adhesive. Let freeze inside the cryotome.CRITICAL STEP: the brain should be oriented straight and parallel to the blade in order to ensure correct slicing.If sections are used as serial sections prepare six tubes for a half brain, or if they will be stored as regions of interest label the tubes accordingly and fill them up with 1.7 mL of cryoprotection solution. Prepare cryoprotection solution by mixing 150 g of sucrose, 200 mL of 1X PBS, 150 mL of ethylene glycol, and complete to a final volume of 500 mL with 1X PBS. Store at 4ºC.Position the metallic holder into the cryotome. Start trimming the OCT adhesive until the brain appears. Start slicing the brain with a thickness of 30 µm, or as desired. Select each slice, submerge in PBS and alternate the storage of each slice into different tubes. This will ensure that a representation of the whole brain is stored on each tube.STOP POINT: Slices can be stored at 4ºC for as long as needed before mounting.

For RNA analysis, prepare a tube containing Trizol or a lysis buffer with an RNAase inhibitor of preference3.For protein extraction, prepare extraction buffer in a 50 mL conical tube containing 1 mM AESBF (or other appropriate proteinase inhibitors), 1 tablet of cOmplete and 2 mL of RIPA buffer and dilute 1:25 with 1X PBS.Place half brain inside a clean Dounce homogenizer and homogenize the brain with 500 uL of extraction buffer. Use small strokes to avoid bubbles. A total of 20-30 strokes should be enough to homogenize the tissue. With a pipette, transfer the homogenized tissue to an Eppendorf and keep on ice. Wash the homogenizer with MiliQ water between samples. Sonicate tubes on water for 10 minutes. Transfer samples to a 4ºC centrifuge, and centrifuge at 14000 rpm (table top centrifuge) for 30 min. Transfer the supernatant to a new tube and if desired, the pellet can be stored for further processing. If extracting protein or RNA from cerebral microvessels is of interest, follow this detailed protocol on extracting cerebral microvessels from cortex4 .After sample preparation:·     Proceed with preferred protocol for protein measurement (Figure 2). We use Pierce BCA Protein Assay Kit (ThermoFisher CAT No. 23225)·     If performing RNA extraction, process samples as quickly as possible and clean all surfaces with RNAzap (ThermoFisher, CAT No. AM9780). Proceed with preferred protocol for RNA extraction. (Figure 3) CRITICAL STEP: The extraction and sectioning of the brain has to be performed as quickly as possible in order to ensure the least amount of RNA or protein degradation.

After perfusion with 1X PBS, the brain through the cranial window should look pale. Carefully remove the cranial window with forceps. Decapitate with scissors, then cut the muscles of the neck and the skin that covers the cranium. Insert the scissors through the foramen magnum, and gently start cutting the cranial bone, up to the rostral side.CAUTION: Take care to not damage the brain during this process.With forceps, remove the lateral bone flaps and expose the brain completely. Slide curved narrow forceps under the brain and carefully tilt it upward. When the brain is sufficiently loose, slide it out of the cranium with the help of the forceps and add to a clean petri dish sitting in an ice bath and wash the brain with ice cold 1X PBS to remove all hairs or other debris. With a clean razor blade, section the brain in half along the sagittal axis. With the help of curved forceps, carefully separate the cortex from the hippocampus (for more details see5) or dissect other brain regions are desired.Immerse one half (for immunostaining) in a tube containing 4% PFA and fix overnight at 4°C. After 24 hours, transfer the brain to a solution containing 30% sucrose. The other half or remaining brain sections can be snap freeze in liquid nitrogen or processed immediately for RNA or protein extraction.STOP POINT: If samples cannot be immediately processed, we recommend homogenizing the tissue immediately using a Dounce homogenizer (or other methods, such as bead mill) in the appropriate buffer, making sure to add RNAase or protease inhibitors depending on need, and then snap freeze and store at -80ºC.

Place the mouse on its back on a grate sitting on top of the rectangular bucket and tape the forelimbs and hindlimbs firmly to the grate. Perform a cut from below the ribs to the upper chest in order to expose the chest cavity. Attach hemostats to the ribs and retract in order to fully expose the heart. Insert a 21G needle attached to a perfusion pump containing 1X PBS into the left ventricle, and then perform a small cut in the right atrium. Start to pump 1X PBS to the mouse, until the fluid runs clear out of the cut in the right atrium. The liver changing from a deep red to a paler shade is an indicator of a good perfusion.CRITICAL STEP: further perfusion with 4% PFA should be avoided as half of the brain will be used for protein extraction and immunostaining results have shown significant improvement when skipping this step. 

The extraction of blood is performed via cardiac puncture. Scruff the mouse from the back with the body hanging straight vertically. Insert a 22-25G needle with a 3 mL syringe just under the ribs at the body centerline. The needle should hit the heart approximately at the level of the elbow (Figure 1). Gently, withdraw the syringe plunger until blood starts to come out. If blood doesn’t come out, move the syringe slightly upwards or downwards until blood is readily withdrawn. A volume of 1-2 mL can be extracted with this technique. Do not apply additional back pressure until the blood has filled the syringe1,2.CRITICAL STEP: proper mouse restrain is important to ensure successful blood collection. This step needs to be done as fast as possible to ensure that vessels won’t collapse before the transcardial perfusion. CAUTION: applying too much back pressure can collapse the heart muscle thus interfering with the blood extraction.Rapidly transfer the blood to a vacutainer blood collection tube with EDTA. If the blood will be used for RNA extraction, apply three parts of Trizol LS per one part of blood, and process immediately or snap freeze for later processing. If the blood will sit for more than a minute in the syringe before transferring to a container with EDTA, then it may be advisable to add some EDTA to the syringe to coat the inside.STOP POINT: samples can be stored at -80ºC until further processing or can be processed fresh. Proceed to extract RNA with a preferred method.

Fractionation of Ovarian Follicles and in vitro Oocyte Maturation and Ovulation Assay of Ciona intestinalis Type A

Ultrasound-based minimally invasive autopsies (MIA/US) is an inexpensive procedure to obtain tissue samples of several organs and, at the same time, reduce the risks of the autopsy procedure in situations of high contagiousness