eamag/protocols-with-steps huggingface dataset:
TITLE: Ligation
AUTHORS: Brian    Teague

[DESCRIPTION]
A DNA ligase is an enzyme that forms phosphodiester bonds -- it can "glue" together two pieces of DNA. The ligase we're using comes from the T4 bacteriophage virus.

Why do we need to do a ligation anyway? Remember, we're going to use a Cas9 protein to "cut" your target gene in a yeast cell. We can program the Cas9 to cut a particular place using a "guide RNA." However, does Saccharomyces cerevisiae -- brewers' yeast -- make Cas9 or a guide RNA on its own? No! So we need to give the yeast cell a set of instructions for making the Cas9 protein and the guide RNA.

We do so by encoding those instructions on a plasmid -- a small circular piece of DNA -- and then putting that plasmid into the yeast cell. Then, the yeast cell "follows" the instructions to make the Cas9 protein and guide RNA. The plasmid we're making has several functional DNA sequences on it -- a diagram is below.

  

The functional sequences on the plasmid are denoted by colored boxes -- you can ignore most of them for now. Note, however, the boxes labelled Cas9 and Guide RNA -- these are the instructions for making Cas9 and the guide RNA. Remember, though, that each group is knocking out a different gene -- so each group needs a different plasmid!

The part that needs to be customized for each group is the "target sequence" in the above diagram. Your instructor started with a plasmid similar to the one above, but where the target sequence is instead replaced by gene for a green fluorescent protein (GFP) -- cells that express it turn bright green! They have cut out the GFP, and now you need to replace it with a targeting sequence for your gene, as in the diagram below.

   

One last thing. Where is your targeting sequence coming from? It's the annealed oligonucleotides from last time. You'll mix the plasmid backbone, your annealed oligos, the ligase enzyme, and a ligase buffer (which contains all of the chemicals the ligase needs to operate), and then the ligation will incubate at room temperature while the enzyme does its thing.

[BEFORE_START]
This protocol depends on the successful completion of the Annealing Oligonucleotides protocol, below:
 


[GUIDELINES]
This protocol involves pipetting very small quantities of liquid. Pipette carefully!

[STEPS]
SECTION: Ligation
1. Dilute the annealed oligonucleotides in   to make 100 µL of working stock at a final concentration of 200 nanomolar (nM)

SECTION: Ligation
2. In the PCR tube, mix in order:
4 µL   
2 µL  of the L2-01 DNA plasmid backbone
 2 µL of the diluted annealed oligos
1 µL   
1 µL  

SECTION: Ligation
3. Flick the tube several times to mix the components, then spin briefly in the microcentrifuge to collect everything at the bottom of the tube.
SECTION: Ligation
4. Incubate at room temperature at least 30 min, and up to 120 min (longer is better).

SECTION: Ligation
5. Store at -20 °C
TITLE: Lentivirus production for primary neuron transduction
AUTHORS: Miguel Da Silva Padilha, Irina Dudanova, Itika Saha, F. Ulrich Hartl, Mark S. Hipp

[DESCRIPTION]
This protocol describes the production of lentiviruses to transduce mouse primary neurons and has to be performed in a biosafety level 2 laboratory

[STEPS]
1. Expand HEK293T cells (LentiX 293T cell line, Takara) for lentiviral packaging to 70-85% confluency in DMEM Glutamax (+4.5 g/L D-glucose, - pyruvate) supplemented with 10% Fetal Bovine Serum (FBS)(Sigma), 1% G418 (Gibco), 1% Non-Essential Amino Acids (Thermo Fisher) and 1% HEPES (Biomol). 
 

2. For lentiviral production, plate cells (~ 4.8 x 106 ) in a three-layered 525 cm2 flask (Falcon).
3. On the following day, transfect cells with 59.52 μg expression plasmid, 35.2 μg packaging plasmid psPAX2 (RRID:Addgene_12260) and 20.48 μg envelope plasmid pVsVg (gift from Dieter Edbauer) using 345.6 μl TransIT-Lenti transfection reagent (Mirus) in 9.6 mL DMEM without FBS.
4. Incubate transfection mix for 20 min at room temperature and exchange the cell medium in the meantime.
5. Add 10 mL of transfection mix to the flask, followed by incubation overnight.
6. Exchange the medium on the following day.
7. After 48-52 h, collect culture medium containing the viral particles and centrifuge for 10 min at 1,200 x g.
8. Filter the supernatant through 0.45 μm pore size filters using 50 mL syringes and add 20 mL Lenti-X concentrator (Takara) to filtered supernatant.
9. Incubate overnight at 4 °C and centrifuge samples at 1,500 x g for 45 min at 4 °C.
10. Remove the supernatant and resuspend the lentivirus pellet in 150 μL TBS-5 buffer (50 mM Tris-HCl pH 7.8, 130 mM NaCl, 10 mM KCl, 5 mM MgCl2).
11. After aliquoting, store lentivirus at -80 °C.
12. Thaw virus preparation immediately before adding to freshly prepared neuronal culture medium.
13. Remove a fifth of the medium from cultured neurons and add the equivalent volume of virus-containing medium.
TITLE: Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR & DNA Cleanup Kit (5 ?g) (NEB #T1030)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (</span><a href="https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEB #T1030</span></a><span>)</span></div></div>

[STEPS]
?. Add 100 μl DNA Cleanup Binding Buffer to the 50 μl sample.
[DNA Cleanup Binding Buffer]
We recommend a sample volume of 50 μl. For smaller samples, adjust the volume with nuclease-free water.
?. Add 300 μl ethanol (≥ 95%). Mix well by pipetting up and down or flicking the tube. Do not vortex.
[Ethanol (≥ 95%)]
?. Insert column into collection tube and load sample onto column.
?. Spin for 1 minute, then discard flow-through.
[Spinning]
?. Re-insert column into collection tube.
?. Add 500 μl DNA Wash Buffer and spin for 1 minute.
[DNA Wash Buffer]
[Spinning]
?. Discard flow-through.
?. Repeat steps 5-7 (Optional). Recommended for removal of enzymes that may interfere with downstream applications (e.g., Proteinase K).
[Repeating steps 5-7]
?. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
[Re-spinning]
?. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix.
[DNA Elution Buffer]
?. Wait for 1 minute, then spin for 1 minute to elute DNA.
[Waiting]
[Spinning]
Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated.
TITLE: NEBNext Ultra II FS DNA Module E7810
AUTHORS: New England  Biolabs

[DESCRIPTION]
<p>The NEBNext Ultra II FS DNA Module contains the enzymes and buffers required to convert a broad range of input amounts of intact DNA into fragmented DNA with 5´ phosphorylated 3´ dA-tailed ends. The module is optimized for use with the NEBNext Ultra II Ligation Module (NEB #E7595) and with the NEBNext Ultra II Q5 Master Mix (NEB #M0544) if amplification is required. The fast, user-friendly workflow has minimal hands on time.</p>
<p> </p>
<p><strong>Note: The Ultra II FS Module is not compatible with bisulfite conversion workflows</strong></p>

[STEPS]
?. [Fragmentation/End Prep]
Fragmentation occurs during the 37°C incubation step. Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. See Figure 1 for a typical fragmentation pattern.  ABC1Fragmentation SizeIncubation @ 37°COptimization2100 bp–250 bp30 min30–40 min3150 bp–350 bp20 min20–30 min4200 bp–450 bp15 min15–20 min5300 bp–700 bp10 min5–15 min6500 bp–1 kb5 min5–10 minFigure 1: Example of size distribution on a Bioanalyzer®. Human DNA (NA19240) was fragmented for 5–40 min.
?. [Fragmentation/End Prep]
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
?. [Fragmentation/End Prep]
Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.
It is important to vortex the enzyme mix prior to use for optimal performance.
?. [Fragmentation/End Prep]
Add the following components to a 0.2 ml thin wall PCR tube on ice:   AB1ComponentVolume per One Library2DNA26 µl3NEBNext Ultra II FS Reaction Buffer7 µl4NEBNext Ultra II FS Enzyme Mix2 µl5Total Volume35 µl
?. [Fragmentation/End Prep]
Vortex the reaction for  and briefly spin in a microcentrifuge.
?. [Fragmentation/End Prep]
In a thermocylcer, with the heated lid set to 75°C, run the following program:5–30 min @ 37°C30 min @ 65°CHold @ 4°C
If necessary, samples can be stores at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation using the NEBNext Ultra II Ligation Module (NEB #E7595) before stopping.
TITLE: Synthesis and preparation of DOPA pheomelanin and eumelanin
AUTHORS: Kazumasa Wakamatsu

[DESCRIPTION]
This is the protocol for synthesizing and preparing DOPA pheomelanin and DOPA eumelanin using tyrosinase.

[STEPS]
1. As precursor(s) of melanin, 1) l-dopa, 2) DHI, 3) a mixture of DHI and DHICA, 4) DHICA, 5) a mixture of l-dopa and l-cysteine, and 6) 5SCD in the presence of l-dopa is used.
2. To a solution of the precursor(s) (1 mmol) in 0.1 M sodium phosphate buffer, pH 6.8 (98 ml), add a solution of mushroom tyrosinase in the same buffer (2 ml) at 25˚C.
3. Carry out oxidation with vigorous (maximal, but avoiding splashing) stirring in a 500 ml Erlenmeyer flask.
2.1.  The amount of mushroom tyrosinase (Sigma-Aldrich, 4270 U/mg) is either 50,000 U or 100,000 U.
4. Stop oxidation (1-4) at 4 h by adding 6 M HCl (2 ml) to make pH 1 and keep the mixture at 4˚C overnight to ensure complete precipitation of the pigment.
5. Collect the precipitate by centrifugation at 3,000 rpm for 10 min and wash with 0.1 M HCl (40 ml x 3).
6. For the preparation of pheomelanins (5 and 6), acidify the oxidation mixture with acetic acid (3 ml) to make pH 3, and wash the precipitate with 1% acetic acid (40 ml x 3). 
7. Dry the melanin powder by lyophilization and equilibrate with moisture in a desiccator with a saturated CaCl2 solution. 
8. Prior to treatment, suspend the synthetic DOPA pheomelanin (DOPA:Cys = 1:1) and DOPA eumelanin (DOPA:Cys = 1:0) in sterile PBS and sonicate overnight.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Training Requirements</strong></p>
<p><strong> </strong></p>
<p>Able to accurately pipet load gel and dilute tissue samples and use Licor Odyssey Imaging Software</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol enriches holobiont <em>Exaiptasia pallida</em> tissue for <em>Symbiodinium</em>, removing more than 99% of host tissue while maintaining high RNA integrity (RIN &gt; 7). The symbiont-enriched fraction is subsequently lysed via bead beating and rna isolated using the Qiagen RNeasy Mini kit.</p>

[BEFORE_START]
<p>Prior to starting, make the host lysis buffer as follows:</p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<p><strong>Component</strong></p>
</td>
<td>
<p><strong>Final Concentration</strong></p>
</td>
<td>
<p><strong>Amount for 100 ml buffer</strong></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">Guanidinium isothiocyanate (dry)</span></p>
</td>
<td>
<p><span style="font-weight: 400;">4 M</span></p>
</td>
<td>
<p><span style="font-weight: 400;">46.26 g</span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">Sodium citrate (1 M stock soln)</span></p>
</td>
<td>
<p><span style="font-weight: 400;">25 mM</span></p>
</td>
<td>
<p><span style="font-weight: 400;">2.5 ml</span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">Sarcosyl (10% stock soln)</span></p>
</td>
<td>
<p><span style="font-weight: 400;">0.5%</span></p>
</td>
<td>
<p><span style="font-weight: 400;">5 ml</span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">Nuclease-free water</span></p>
</td>
<td> </td>
<td>
<p><span style="font-weight: 400;">To 100 ml total volume</span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Add 2-mercaptoethanol to a total concentration of 1% (v/v) to the volume of buffer that will be used within 24 hours. Prior to the addition of 2-mercaptoethanol, the buffer is stable at room temperature.</p>

[GUIDELINES]
<p>This protocol was developed using small (~0.2 mm oral disc) anemones for extraction. With anemones hosting heterologous symbionts at low density (e.g. <em>S. trenchii</em>), pooling multiple anemones for each extraction is useful for increasing yield.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Cloning with Golden Gate
AUTHORS: Raining Wang, Melinda Wheelock

[DESCRIPTION]
This protocol details the cloning and transformation.

[STEPS]
SECTION: Wild-type target assembly
1. Golden Gate assembly:

If the wild-type insert is obtained from PCR amplification, ensure the PCR product is cleaned.
 

SECTION: Wild-type target assembly
2. Made additional digestion mix.
 
 
 
SECTION: Wild-type target assembly
2.1. Aliquot 5 µL of digestion mix to each of the golden gate reactions. 

SECTION: Wild-type target assembly
2.2. Set the thermal cycler for the program below: 
 
 
 

SECTION: Wild-type target assembly
1.1. A negative control reaction with H2O instead of insert is recommended. 
SECTION: Wild-type target assembly
1.2. Set the thermal cycler for the program below:
 
 
 
 

SECTION: Wild-type target assembly
3. Purify each reaction with Zymo Clean & Concentrate -5 kit.
SECTION: Wild-type target assembly
4. Transformation:

Add 1 µL-2 µLof DNA from sample reaction and negative control to chemical competent E. coli cells by following NEB stable competent cell transformation protocol.

 

SECTION: Wild-type target assembly
5. Incubate overnight at 30 °C for 1440 min.

SECTION: Wild-type target assembly
4.1. Consider to add Puc 19 and H2O as controls to the transformation process.
SECTION: Wild-type target assembly
6. Colony selection and mini prep: 

Check negative control plate to ensure the background colony is less than 10% of the colony counts from the sample plate.
SECTION: Wild-type target assembly
7. Pick 2-3 colonies for an overnight growth in 3 mLof LB + amp (1:1000) per colony for mini prep.

SECTION: Wild-type target assembly
8. Sequence confirmation by nanopore sequencing.
TITLE: Imaging Mass Cytometry Compensation Slide Acquisition
AUTHORS: Michelle Daniel, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP describes the slide acquisition for spill-over correction of Imaging Mass</div><div class = "text-block">Cytometry experiments. The protocol makes use of the compensation slide, which was</div><div class = "text-block">prepared previously following the SOP:</div><div class = "text-block">“HuBMAP_Protocol_3_Compensation_Slide_Preparation.pdf” to measure each</div><div class = "text-block">conjugated antibody individually and to record all observed spill-over. The resulting</div><div class = "text-block">compensation matrix can be used to compensate data that has been recorded using the</div><div class = "text-block">same antibody conjugates.</div></div>

[STEPS]
?. [Set-Up]
1. Attach the flat-bed scanner to a computer and take an image of the compensation slide.2. Create a folder on the CyTOF computer on the “E” drive.3. Save the image of the compensation slide in that folder with the same name as the compensationslide.4. Insert the compensation slide into the Hyperion.5. Under Data Acquisition create a new File (“Empty12AF”).6. Import the slide image.7. Save the mcd file in the newly created folder.8. Create a panorama across all the spots. Potentially, the spots may be hard to see in the actualcamera view.
Requires a tuned and running IMC. For tuning see: dx.doi.org/10.17504/protocols.io.bf2gjqbw
?. [Create ROIs]
1. For each of the spots, create an ROI with the size of 10x200 (10 lines with 200 pixels).2. Name each ROI with the metal name of the conjugate (e.g. “Gd156”, “Ir191” etc).3. Apply a panel with the same (or more) channels and settings as measured or will be measuredfor the experiment to be compensated. When creating the panel, make sure to actually select thecorrect metal isotopes as this is important for the spill-over matrix generation.4. Check “Generate Text File”.5. Hit Run.
TITLE: Protocols for " Efficient and stable metabarcoding sequencing data using DNBSEQ-G400 sequencer validated by comprehensive community analyses"
AUTHORS: Xiaohuan Sun, Yuehua  Hu, Zewei  Song

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sequencing in DNBSEQ-G400 of this study was carried out in BGI-Shenzhen. Amplicons from 1276 soil samples were randomly and equally distributed into 3 DNBSEQ runs for both 2×200 paired-end (PE200) and 400 bases single-end sequencing (SE400). The left libraries of total 1276 samples were repetitively sequenced two more times by SE400 using different DNBSEQ-G400 machines. Three randomly chosen soil samples from each forest plot (ALS268, LJ105 and NBH217) were all amplified 3 times separately as PCR replicates. The corresponding 9 sequencing libraries were sequenced repetitively by 3 DNBSEQ runs. </div><div class = "text-block">Sequencing was carried out using the entire lane of the Illumina MiSeq platform in the University of Minnesota Genomic Center. Amplicons of the 1276 forest soil were normalized and pooled randomly in equal molar ratios into 8 parallel sequencing runs on MiSeq platform. Again, three randomly chosen soil samples from each forest plot (ALS268, LJ105 and NBH217) were all amplified 3 times separately as PCR replicates. The corresponding 9 sequencing libraries were sequenced repetitively by 8 MiSeq runs. </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/cypjbgx9x.jpg" /></div></div>

[STEPS]
TITLE: Preparation of ATP13A2 microsomes from Sf9 cells
AUTHORS: Sue  Sim, eunyong_park  

[DESCRIPTION]
Isolate microsomes from Sf9 cells expressing ATP13A2

[STEPS]
1. Thaw Sf9 cell pellets at room temperature (typical size around 5g for 0.4 L of culture)
2. All subsequent steps should be carried out at 4 °C 

3. Wash pellet twice with 15 mL of Phosphate-buffered Saline, centrifuge at 1000 x g, 7 min, 4 °C in between washes

3.1. Gently resuspend by inverting tube and pipetting
4. Collect cells after final wash by centrifugation at 1500 x g, 7 min, 4 °C 

5. Resuspend cells in 10 mL Lysis Buffer
6. Swell cells in Lysis Buffer by incubating on ice for 10 min to 15 min 

7. Lyse with Dounce homogenizer, 40 strokes tight
8. Dilute homogenate in equal volume Resuspension Buffer and mix
9. Further lyse with Dounce homogenizer, 20 strokes tight
10. Spin down at 1000 x g, 10 min, 4 °C to remove nuclear fraction and unbroken cells and save supernatant

11. Spin down at 10000 x g, 20 min, 4 °C (Sorvall SS-34 rotor) to remove mitochondrial-lysosomal fraction and save supernatant

12. Transfer supernatant to ultracentrifuge tubes and spin down at 200000 x g, 35 min, 4 °C (Beckman Type 45 Ti rotor) to collect microsomes

13. Resuspend microsomal pellet in Storage Buffer
14. Measure microsome concentrations based on total protein concentration using the Bradford Assay and bovine serum albumin as a standard
15. Flash-freeze in aliquots of 25-100 μL at concentrations between 2-5 mg/mL using liquid nitrogen and store at -80C until use
TITLE: Transcardial Perfusion in Mouse
AUTHORS: Michael Henderson

[DESCRIPTION]
This protocol details about the transcardial perfusion in mouse.

[STEPS]
SECTION: Transcardial Perfusion
1. Make mixture of ketamine:xylazine:acepromazine (4:2:1) sufficient for anesthesia of all mice (~30 µL/ 20 gmouse). Record ketamine used in controlled substance log book.
SECTION: Transcardial Perfusion
2. Apply anesthesia to one mouse via intraperitoneal injection, and place mouse in bucket long enough for anesthesia to take effect. Apply a hard toe pinch until mouse no longer reacts, ensuring that the mouse can no longer feel pain before proceeding.
SECTION: Transcardial Perfusion
3. Place mouse, abdomen-up, on Styrofoam block wrapped in lab mat. Spray mouse abdomen with 70% ethanol. Grasp skin below ribcage with forceps and cut skin with scissors from middle up either side towards the armpits, cutting through ribcage.
 

SECTION: Transcardial Perfusion
4. Remove pericardium and peripheral fat to expose heart. Hold back ribs with hemostats. 
 

SECTION: Transcardial Perfusion
5. Place blunted butterfly needle into left ventricle and cut right atrium. Push PBS through syringe by using perfusion pump at 2 mL/minute rate.
 

SECTION: Transcardial Perfusion
6. After perfusion is complete, remove brain, spinal cord and other desired parts and transfer to fixative. 
 

  

SECTION: Perfusion Solutions
7. Weigh PFA under the hood and using bench pads.
SECTION: Perfusion Solutions
8. Heat and stir (in chemical hood).
SECTION: Perfusion Solutions
9. Lets dissolve the PFA around 60 °C (use the thermometer). Do not let the temperature exceed 65 °C(remove from hot plate temporarily if necessary).


SECTION: Perfusion Solutions
10. Once dissolved but still foggy, pH 7.4 with NaOH (it should make the PFA clear).

SECTION: Perfusion Solutions
11. Prepare a big filter funnel using Whatman filter paper.
SECTION: Perfusion Solutions
12. Filter the 4% PFA into a liter bottle/w lid.
SECTION: Perfusion Solutions
13. Store the PFA covered at4 °C.
TITLE: Unsucessful atemps on transfromation of the Icthyosporean Sphaeroforma artica
AUTHORS: Elena Casacuberta, Cristina Aresté, Omaya Dudin , Elena Casacuberta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Testing electroporation on Spaheroforma artica with two different electroporators</div><div class = "text-block">Neon (invitrogen)</div><div class = "text-block">Lonza (nucleofector)</div></div>

[STEPS]
?. Cells were seeded 24 hours prior transfection
?. 200.000 cells per transfecion condition were tested
?. Tested techniques and settings are as follows (with 1 to 5 micrograms of DNA endogenous promoter (actin) plus fluorescent reporter (venus or mCherry)   ABCD1S.arcticaNeon1000-2500V, 10-40 ms, 1-3 pulsesn/a (not successful)2&nbsp;Lipofectamina&nbsp;n/a (not successful)3&nbsp;LONZA®16 preset codes P3/P4/P5 buffern/a (not successful)
ABCD1S.arcticaNeon1000-2500V, 10-40 ms, 1-3 pulsesn/a (not successful)2&nbsp;Lipofectamina&nbsp;n/a (not successful)3&nbsp;LONZA®16 preset codes P3/P4/P5 buffern/a (not successful)
?. cells were recovered inmediately in MB medium and inspected for positive transfection by fluorescence after 24, 48 and up to 5 days.
TITLE: Rodent brain processing for histological analyses (update)
AUTHORS: Miquel Vila

[DESCRIPTION]
Protocol for rat brain processing in order to perform histological analyses

[STEPS]
SECTION: Rat perfusion
1. Deeply anesthetize animals with sodium pentobarbital (50 mg/kg, i.p.)
SECTION: Rat perfusion
2. Perfuse through the left ventricle with saline [0.9% (wt/vol)] at room temperature (RT)
SECTION: Rat perfusion
3. Perfuse again with ice-cold formaldehyde solution 4% in PBS buffered for histology

SECTION: Postfixation
4. Remove brains and post-fix them for 24 h in the same fixative
SECTION: Processing for microtome sectioning
5. Wash twice with 0.1 M PBS and process for paraffin embedding following standard procedures (performed by an external facility) 

Alternatively, brains could be stored in 0.1 M PBS at 4 °C (not for over a month without changing the PBS) prior paraffin processing
SECTION: Processing for cryostat sectioning
6. Cryoprotect for 24-48 h (until they sink) in 30% sucrose at 4 °C
SECTION: Sectioning
10. Perform sectioning with a sliding microtome at 5-μm-thickness for paraffin samples or
in a cryostat at 20- or 30-μm-thickness for frozen samples. 
SECTION: Processing for cryostat sectioning
7. Exchange sucrose for 0.1 M PBS
SECTION: Processing for cryostat sectioning
8. Immerse brains in cold (-30ºC) 2-methylbutane for 30 s and store at -80 °C
SECTION: Processing for cryostat sectioning
9. Include in OCT