eamag/protocols-with-steps huggingface dataset:
TITLE: TissueCyte Image QC
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the QC step that occurs after the week of TissueCyte runs, and prior to analysis.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. </span></div></div>

[STEPS]
TITLE: UC Davis - Hematoxylin and Eosin (H&E)
AUTHORS: Jennifer  Rutkowsky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Staining tissues on a slide with Heatoxylin and Eosin.</div><div class = "text-block"><span style = "font-weight:bold;">Modified from: </span><span>H &E protocol, UC Davis Clinical Pathology lab</span></div></div>

[STEPS]
?. Heat slide to 37ºC for 20 min
?. Move through the following solutions sequentially for the noted amount of time
?. Mount tissue with a coverslip
TITLE: Marchantia Cryopreservation of Gemmae
AUTHORS: Marius Rebmann, marta tomaselli, Linda Silvestri, Susana Sauret-Gueto, Michelle Lim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Simplified cryopreservation protocol for </span><span style = "font-style:italic;">Marchantia polymorpha </span><span>gemmae, based on (</span><a href="https://doi.org/10.1093/pcp/pcv173" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Tanaka et al. 2016, Plant and Cell Physiology</span></a><span>). Enables long term storage of gemmae at -80°C. </span></div></div>

[STEPS]
?. [Preculture]
Collect fresh gemmae and plate on preculture plates, incubate 1-3 days under normal growth conditions (e.g. 21°C constant light)
?. [Encapsulation]
Place small drops (approx. 40μL) of alginate solution on an empty 4.5cm petridish/multi-well plate to form beads.
?. [Encapsulation]
Use forceps to transfer gemmae from preculture plates, place 1-5 gemmae inside each bead. Proceed quickly to step 4 to avoid gemmae sinking to the bottom or edge of the bead.
?. [Encapsulation]
Add a drop of CaCl2 solution to each bead, make sure to not displace gemmae from the bead.
?. [Encapsulation]
Allow beads to solidify for 10min.
?. [Encapsulation]
Use a serological pipette to fill the plate with loading solution, submerging the beads. Saok beads for 30min.
?. [Encapsulation]
Use a serological pipette to remove the loading solution without disrupting the beads.
?. [Encapsulation]
Remove lid and air dry beads in flowhood for >2h.
?. [Encapsulation]
Use forceps or scalpels to transfer beads into 1.5mL Eppendorf tubes (max. 10 beads/tube).
?. [Encapsulation]
Flash freeze tubes in liquid nitrogen for >2 minutes.
?. [Encapsulation]
Move tubes to -80°C for long term storage.
?. [Thawing and Recovery]
Remove tubes from freezer and immediately place in a 37ºC water bath or heat block for 2 minutes.
?. [Thawing and Recovery]
Add 1.5mL thawing solution to each tube, incubate at room temperature for 10 minutes.
?. [Thawing and Recovery]
Replace thawing solution with rinse solution. Incubate at room temperature for 10 minutes.
?. [Thawing and Recovery]
Remove rinse solution, transfer beads on 1/2 Gamborg plates, incubate under normal growth conditions (e.g. 21°C constant light) until thalli grow
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to use agarose with a low gelling point (27-30°C; often called low-melting agarose) to immobilize Aiptasia larvae for microscopy.</p>
<p>Larvae stay alive and in one place for prolonged periods of several hours but continue to mainly spin around the longitudal axis. After imaging, larvae can easily be retrieved from the agarose and transferred back to artificial sea water.</p>
<p> </p>
<p> </p>

[BEFORE_START]
<p><u>Preparation of the agarose:</u></p>
<ol>
<li><strong>Weight 1 g low melting agarose into 50 ml ASW (2% w/v) in a small glass flask.</strong></li>
</ol>
<p><em>Using higher concentration (up to 8%) makes the agarose harder to handle but doesn’t improve the embedding notably (at least in my experience).</em></p>
<ol start="2">
<li><strong>Heat the mix without lid in a microwave to completely dissolve the agarose.</strong></li>
<li><strong>Transfer 1 ml of the agarose into a sterile microcentrifuge tube, pour the rest into a sterile 50 ml tube.</strong></li>
<li><strong>You can let the agarose cool down and solidify.</strong></li>
</ol>

[GUIDELINES]
<p>A protocol for making the Valap for sealing the sample can be found under the following link: <a href="http://cshprotocols.cshlp.org/content/2010/12/pdb.rec12380" target="_blank" rel="noopener noreferrer">http://cshprotocols.cshlp.org/content/2010/12/pdb.rec12380</a></p>
<p>Valap is basically a wax-like substance which has the advantages over other ways of sealing (for example nail-polish) that it is hydrophobic and doesn't contain any solvents which might mix with the aqueous sample and interfere with viability and fluorescence.</p>
<p> </p>
<p><em>We are actually using a device with a small resistance wire loop to take a small amount of cold, solid Valap and then to melt it directly at the slide-mounted cover slip.</em></p>

[STEPS]
?. 
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?. 
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?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
?.
TITLE: Picking worms
AUTHORS: Bonnie  Evans

[DESCRIPTION]
Transferring worms using a platinum wire pick

[STEPS]
1. Light the ethanol burner. Take care that there is nothing directly above it, eg. papers attached to shelf. 
2. Place the platinum wire part of the pick in the flame to sterilise it. Let it cool before touching the plate. 
3. Pick up some bacteria from the plate with the platinum wire. 
4. Look down the microscope to identify the worm you want to pick (e.g for L4 worms, look for a white semi-circle in the worm mid-section). Gently touch the worm with the bacteria to pick it up. 
5. Transfer the worm to the new plate by gently placing the pick on the agar and allowing it to crawl out of the bacteria on the wire. Be careful not to penetrate the agar.
TITLE: Extraction of bacterial DNA using ChargeSwitch gDNA Mini Bacteria kit on KingFisher Flex robot
AUTHORS: Leyi  Wang, Carol Maddox, Melanie  Prarat, Yan  Zhang, Lifang Yan, Akhilesh Ramachandran, Sai Sankara Narayanan, Girish Patil, Sarah  Nemser, Mothomang  Oyinloye, Olgica  Ceric

[DESCRIPTION]
This procedure is used to extract genome DNA of bacteria isolates using the ChargeSwitch gDNA Mini Bacteria kit on the KingFisher Flex Robot. The process consists of Sample processing, Prepare plates for Robot, and Operate on KingFisher Flex machine.

[BEFORE_START]
At the first time use, add RNase A to Resuspension Buffer (R4).

[GUIDELINES]
In the whole procedure, need wear PPE

[STEPS]
1.1. 
Bacterial Broth Culture

If using broth, pick single colony and grow in 5 ml Trypticase Soy Broth (TSB), overnight grow at 37 degree without shaking. Use 0.4 ml of the bacterial culture and centrifuge for 4min at 8000 rpm, discard supernatant and resuspend the cell pellet in 100 μL of Resuspension Buffer (R4) containing RNase A, then add 5 ul of lysozyme solution (50 mg/ML). Mix by vortexing. After adding RNase A to Resuspension Buffer (R4), store the buffer at 4 degrees Celsius. 
1. Sample processing
1.2. Bacterial Colonies in Plate

If using bacterial colonies, scoop 3 colonies into 100 μL of Resuspension Buffer (R4) containing RNase A, and then add 5 ul of lysozyme solution (50 mg/ML). Mix by vortexing.
2. Prepare plates for Robot
2.1. Load all resuspension solution prepared in 1.1. or 1.2  into 2.4 mL 96 deep well Sample plate.
2.2. Prepare Elution Plate by adding 100 ul of Elution buffer to each well of the 0.5ml 96-well plate.
2.3. Prepare 3 Wash Plates (Wash Plate 1, Wash Plate 2 and Wash Plate 3) by adding 650 ul Wash Buffer (W12) to each well of 2.4 mL 96 deep well plates.
3. Operate on KingFisher Flex machine
3.1. Install script of ChargeSwitch gDNA Mini Bacteria kit on KingFisher Flex by requesting the script through ThermoFisher and installing it using BindIt software
3.2. Select the right script/method and then click start. 
3.3. Load 0.5ml 96-well plate containing the Tip comb.
3.4. Load Elution plate.
3.5. Load Wash Plate 1, Wash Plate 2, and Wash Plate 3 one by one.
3.6. Load Sample plate. The instrument will automatically start processing the sample plate.

(While the instrument is processing the sample plate, prepare a premix of 500 ul Lysisi Buffer (L14) and 10 ul of Proteinase K solution).
3.7. 6)	After 10 mins of sample processing, there is the first pause. Once the Instrument pauses, pick up the sample plate from the instrument and add 500 ul of the premix prepared in step 5 (500 ul Lysis Buffer/Proteinase K mixture) to each sample well of Sample plate.
3.8. Then load Sample plate back into the instrument and click Start.
3.9. After 10 mins, there is a second pause, take the sample plate off the instrument and add 40 ul of Charge Switch Magnetic Beads and 300 ul Binding Buffer (B8) to each sample well, and then load the Sample plate back into the instrumentand click Start. The machine will do the remaining steps until it is done.
TITLE: Ambulatory Recordings of Wireless Bladder and Colon Devices in Pigs
AUTHORS: Brett Hanzlicek

[DESCRIPTION]
This protocol is for collecting ambulatory, wireless data from a coloMOCA impantled into a pig colon.


[STEPS]
SECTION: Ambulatory Testing
1. Place BluMOCA receiver radio into pocket on the pig jacket.
SECTION: Ambulatory Testing
2. Wake the coloMOCA with the waker set between 9V-15V, 1.5A.
SECTION: Ambulatory Testing
3. Plug the Bluetooth dongle into the computer.
SECTION: Ambulatory Testing
4. Record from ColoMOCA using LabVIEW software; take notes about pig movement, eating, bowl movement etc.
SECTION: Ambulatory Testing
5. Turn off ColoMOCA with waker.
SECTION: Ambulatory Testing
6. Remove BluMOCA receiver.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Determine number of samples <strong>and</strong> standards to test in 96 well plate format.  Multiply by 2 if running everything in duplicate for total number of wells.  <br /><br />Use a black-walled plate with black bottoms if possible.  Black-sided wells with clear bottoms or white-sided wells will also work, but background will be higher due to reflected fluorescence in the wells.  Do not use clear microtiter plates for fluorescence readings.<br /><br />You will need 100 μl diluted PicoGreen reagent per well.<br /><br />Total amount of 1X TE per assay will be 200 μl per well which includes the amount of TE used to dilute the PicoGreen reagent.

[GUIDELINES]
<table style="width: 392px; height: 566px;">
<tbody>
<tr>
<td><strong>DNA Standard</strong></td>
<td><strong>Vol.μl DNA</strong></td>
<td><strong>Vol.μl 1x TE</strong></td>
<td><strong>Vol μl PicoGreen (1:200)</strong></td>
<td><strong>Final DNA Standard ng/mL</strong></td>
</tr>
<tr>
<td>Low DNA Standard </td>
<td>0</td>
<td>100</td>
<td>100</td>
<td>0</td>
</tr>
<tr>
<td>(1:1000 of 100 μg/ml)</td>
<td>1</td>
<td>99</td>
<td>100</td>
<td>0.5</td>
</tr>
<tr>
<td> </td>
<td>5</td>
<td>95</td>
<td>100</td>
<td>2.5</td>
</tr>
<tr>
<td> </td>
<td>10</td>
<td>90</td>
<td>100</td>
<td>5.0</td>
</tr>
<tr>
<td> </td>
<td>20</td>
<td>80</td>
<td>100</td>
<td>10</td>
</tr>
<tr>
<td> </td>
<td>50</td>
<td>50</td>
<td>100</td>
<td>25</td>
</tr>
<tr>
<td> </td>
<td>80</td>
<td>20</td>
<td>100</td>
<td>40</td>
</tr>
<tr>
<td> </td>
<td>100</td>
<td>0</td>
<td>100</td>
<td>50</td>
</tr>
<tr>
<td>High DNA Standard </td>
<td>0</td>
<td>100</td>
<td>100</td>
<td>0</td>
</tr>
<tr>
<td>  (1:50 of 100 μg/ml)</td>
<td>1</td>
<td>99</td>
<td>100</td>
<td>10</td>
</tr>
<tr>
<td> </td>
<td>5</td>
<td>95</td>
<td>100</td>
<td>50</td>
</tr>
<tr>
<td> </td>
<td>10</td>
<td>90</td>
<td>100</td>
<td>100</td>
</tr>
<tr>
<td> </td>
<td>20</td>
<td>80</td>
<td>100</td>
<td>200</td>
</tr>
<tr>
<td> </td>
<td>50</td>
<td>50</td>
<td>100</td>
<td>500</td>
</tr>
<tr>
<td> </td>
<td>80</td>
<td>20</td>
<td>100</td>
<td>800</td>
</tr>
<tr>
<td> </td>
<td>100</td>
<td>0</td>
<td>100</td>
<td>1000</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
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?.
TITLE: Soluble and insoluble A-SYN fractionation
AUTHORS: michela.deleidi, María José Pérez J., Hariam Raji, Pascale Baden, Federico   Bertoli

[DESCRIPTION]
Soluble/insoluble alpha-synuclein fractionation is a technique used to separate different forms of the alpha-synuclein protein based on their solubility properties.

[STEPS]
SECTION: Soluble and insoluble A-SYN fractionation
1. Perform extraction and detection of Triton-soluble (T-sol) and Triton-insoluble (T-insol) alpha-synuclein as described in Stojkovska and Mazzulli 53. 
SECTION: Soluble and insoluble A-SYN fractionation
2. Lyse individual organoids in 1% Triton X-100 extraction buffer supplemented with 1X PIC, 50 millimolar (mM) NaF, 2 millimolar (mM) NA3VO4 and 0.5 millimolar (mM) PMSF. 

 Extraction buffer
      Triton X-100  1%    NaCl  150 mM    glycerol  10%    HEPES pH 7.4  25 mM    EDTA  1 mM    MgCl2  1.5 mM  
 
SECTION: Soluble and insoluble A-SYN fractionation
3. Homogenize samples with a pestle and incubate on a platform shaker in an ice-water slurry for 30 min, followed by three freeze/thaw cycles and ultracentrifugation at 100000 x g, 30 min, 4 °C. 

SECTION: Soluble and insoluble A-SYN fractionation
4. Collect the supernatant (Triton-X Soluble fraction).
SECTION: Soluble and insoluble A-SYN fractionation
5. Wash the remaining pellet in Triton X-100 extraction buffer followed by another ultracentrifugation at 100000 x g. 

SECTION: Soluble and insoluble A-SYN fractionation
6. Resuspend the pellet in 2% SDS buffer containing 50 millimolar (mM) Tris, pH 7.4 and 1X PIC, boil it for 10 min at 100 °C (Triton-X insoluble Fraction) and label the T-insol fraction. 
SECTION: Soluble and insoluble A-SYN fractionation
7. Sonicate Tx-Insoluble samples for 10 min at 30% power, 20C in a cup horn sonicator (Qsonica-Q700), and then boil them again for 10 min at 100 °C. 

SECTION: Soluble and insoluble A-SYN fractionation
8. Ultracentrifuge Tx-Insoluble samples at 100000 x g, 30 min, 21 °C. 
SECTION: Soluble and insoluble A-SYN fractionation
9. Collect the supernatant (SDS-soluble fraction).
SECTION: Soluble and insoluble A-SYN fractionation
10. Detect protein concentrations using a BCA assay and load 30 µg of total protein for each condition.
TITLE: Western blotting to detect ATP13A2
AUTHORS: Stephanie Vrijsen, Marine Houdou, Peter Vangheluwe

[DESCRIPTION]
Protocol to detect ATP13A2 via Western Blotting.

[STEPS]
SECTION: Harvesting cells
1. Collect the cells by using TrypLE.
SECTION: Harvesting cells
2. Centrifuge cell suspensions at 450 xg, 4°C for 5 min.

SECTION: Harvesting cells
3. Resuspend cell pellets with DPBS and centrifuge following the same indications as in 2. Repeat once.
SECTION: Harvesting cells
4. Discard supernatants and keep cell pellets on ice.
SECTION: Cell lysis and protein concentration determination
5. Resuspend cell pellets in RIPA buffer (RIPA Lysis and Extraction Buffer) supplemented with protease cocktail inhibitors. 
SECTION: Cell lysis and protein concentration determination
6. Vortex 30 s and keep on ice for 10 min.

SECTION: Cell lysis and protein concentration determination
7. Centrifuge at 20,000g, 4 °C for 10 min.

SECTION: Cell lysis and protein concentration determination
8. Keep supernatants on ice to proceed with protein concentration determination using the micro-BCA Protein Assay Kit.
SECTION: SDS-PAGE and Western blot
9. Loading
SECTION: SDS-PAGE and Western blot
9.1. Mix 10 µg of protein with NuPAGE LDS sample buffer and 5% β-mercaptoethanol final.
SECTION: SDS-PAGE and Western blot
9.2. Boil for 5 min at 95 °C

SECTION: SDS-PAGE and Western blot
9.3. Load protein on pre-cast 4-12% Bis-Tris gels. Include at least one lane with a protein ladder.
SECTION: SDS-PAGE and Western blot
10. Running
SECTION: SDS-PAGE and Western blot
10.1. Run for and 80 minat 130V in MES buffer.

SECTION: SDS-PAGE and Western blot
11. Transfer
SECTION: SDS-PAGE and Western blot
11.1. Transfer onto PVDF membranes using a liquid transfer and following settings: 100V, 90 min, 4°C. Use transfer buffer with 10% methanol. 

SECTION: SDS-PAGE and Western blot
12. Blocking
SECTION: SDS-PAGE and Western blot
12.1. 60 min Block membranes with blocking buffer (5% milk powder in 1X TBS and 0.1% Tween20 (REF)) for 60 min at room temperature, 19 rpm.

SECTION: SDS-PAGE and Western blot
13. Primary antibodies
SECTION: SDS-PAGE and Western blot
13.1. Incubate membrane with primary antibodies in solution (1% bovine serum albumin in 1X TBS-Tween20 (TBS-T) buffer), 60 minat 4°C, 19 rpm.

SECTION: SDS-PAGE and Western blot
13.2. Wash membrane three times for 5 min in TBS-T, 19 rpm.

SECTION: SDS-PAGE and Western blot
14. Secondary antibodies
SECTION: SDS-PAGE and Western blot
14.1. Incubate membrane with peroxidase-conjugated secondary antibodies in solution (1% milk powder in 1X TBS-T) for 60 minat room temperature and, 19 rpm.

SECTION: SDS-PAGE and Western blot
14.2. Wash membrane five times for5 minin TBS-T, 19 rpm.

SECTION: SDS-PAGE and Western blot
15. Detection
SECTION: SDS-PAGE and Western blot
16. Use a chemiluminescence reagent to detect signal and acquire with a Biorad Camera (Vilber Lourmat) and its software (ImageLab).
TITLE: Single cell survival assay
AUTHORS: Hanqin  Li, Dirk Hockemeyer

[DESCRIPTION]
This protocol describes the experimental procedure used to measure single cell survival rates post nucleofection of human pluripotent stem cells (hPSCs).

General Notes:
1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

[STEPS]
1. hPSCs are cultured on MEFs as described in the collection "Thawing, Passaging, and Freezing of hPSCs on MEFS" dx.doi.org/10.17504/protocols.io.b4msqu6e
2. 0.5 million hPSCs are nucleofected as described in “Nucleofection of hPSCs" dx.doi.org/10.17504/protocols.io.b4pcqviw
3. Seed 1/100, 1/500 of cells post-nucleofection into one well of a 6-well plate containing hPSCs medium with Rock inhibitor. Prepare at least 2 wells for each seeding density as replicates. 
4. Change medium at day 3, 6, 8, 10, and then daily until colonies grow to medium size.
5. Perform Alkaline phosphatase (AP) staining as described in “Pluripotency markers staining” dx.doi.org/10.17504/protocols.io.b4yyqxxw
6. Image the entire stained well with ChemiDoc (Bio-Rad). Use a white cardboard as background. 
7. Count AP positive colonies, N.
8. Calculate single cell survival rate by: N x dilution factor/500,000. 
      E.g. For the wells with seeding density 1/100, the dilution factor is 100. 
3.1. hPSC medium 
     DMEM/F12      385 ml        Fetal Bovine Serum (FBS)      75 ml        Knockout Serum Replacement      25 ml        L-Glutamine (100X)      5 ml        Penicillin & Streptomycin   (100X)      5 ml        MEM Non-Essential Amino Acids   (100X)      5 ml        2-Mercaptoethanol (10,000X)      50 ul        Heat Stable Recombinant Human   FGF2 (25ug/ml)      80 ul   
 
L-Glutamine (100x)
 


2-Mercaptoethanol (10,000X)
 


Heat Stable Recombinant Human FGF2 (25 µg/ml)
 

hPSCs Medium + Rock inhibitor
 


Y-27632 (1,000X)
TITLE: CPMU
AUTHORS: Bjorn Bartholdy, a.g.henry  

[DESCRIPTION]
Mineralising solution for the oral biofilm. From Sissons et al. 1991.

[BEFORE_START]
This solution MUST be prepared in a sterile and starch-free environment.

[STEPS]
1. Add 300 mL distilled (or deionized) dH2O to a 1000 mL  beaker, with stirring and heat 60 °C 
2. Add:

- 1.55 g   
- 1.44 g   
- 0.72 g   
- 0.08 g   
- 30 g   

3. Add the remaining 700 mL  and keep stirring until precipitate has completely dissolved
Store in fridge at 4 °C
TITLE: OMS Atlas FFPE Spatial Mapping
AUTHORS: Brett   Johnson, Danielle  Galipeau, George  Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections a FFPE block, prepares the resulting slides, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer FFPE blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing. Coordinate execution of this sectioning protocol to allow for shipping within the same business week.

[STEPS]
SECTION: Preparation
1. Verify the identity of the FFPE block to be cut against written request for sectioning.
SECTION: Preparation
2. Label each Tanner slide with a unique BEMS ID and slide number, corresponding to the written request and FFPE spatial map (below).
 

SECTION: Sectioning
3.  Align block on microtome to minimize tissue loss.
SECTION: Sectioning
4. Face into block at 5μm until full section of tissue is achieved.
SECTION: Sectioning
5. Cut adequate ribbon at 5μm to cover all 8 serial sections.
SECTION: Sectioning
6.  Mount tissue sections onto Tanner slides to maintain serial order and orientation of sections.
SECTION: Processing
7.  Perform hematoxylin and eosin (H&E) staining on slides #1 and #8.
8. Bake slides #2 through #7.

8.1. Place slides #2 through #7 in 55 °C oven overnight.
8.2.  The next morning, raise the oven temperature to 65 °C for up to 60 min.
Note: Slides should be baked at 65 °C for at least 30 minutes.

8.3.  Remove slides from oven. Place slides in slide boxes and store at room temperature.
9. Deliver slides #2 through #7 to BioLibrary for distribution.
TITLE: Anatomical Verification (Mendonça et al 2024)
AUTHORS: Marcelo D Mendonça

[DESCRIPTION]
Protocol for anatomical verification (sample prep, immunostaining and imaging)  in Mendonça et al 2024. 

[BEFORE_START]
Animals were euthanized after completion of the behavioural tests.

[STEPS]
SECTION: Sample Preparation
1. Anaesthetize mice with isoflurane, followed by intraperitoneal injection of ketamine-xylazine (5 mg/kg xylazine; 100 mg/kg ketamine). 
SECTION: Sample Preparation
2. Perfuse transcardially in 1% phosphate buffered saline (PBS) and then 4% paraformaldehyde (PFA)  in PBS.
SECTION: Sample Preparation
3. Dissect brains after perfusion is completed. 
SECTION: Sample Preparation
4. Keep brains in 4% PFA solution overnight and then transferred to 1x PBS solution.
SECTION: Sample Preparation
5. Section brains coronally in 30 to 50-um slices (using a Leica vibratome VT1000S) and keep in PBS before mounting or immunostaining. 
SECTION: Immunostaining for TH (Day 1)
6. Rinse 3 times with TBS (10 min each)
SECTION: Imaging
17. Images were taken using a wide-field fluorescence microscope (Zeiss AxioImager). 
SECTION: Immunostaining for TH (Day 1)
7. Peroxides treatment (inhibition peroxidase an permeabilization) 5 min
1.5 mL methanol
450 µL H2O2
13.05 mL TBS
(this step is for DAB staining but we kept it because it favors the penetrance of the Ab)
SECTION: Immunostaining for TH (Day 1)
8. Rinse 3 times with TBS (10 min each)
SECTION: Immunostaining for TH (Day 1)
9. Membrane permeabilization treatment 15min (or 20min for Ab that don’t penetrate well)  
300 µL Triton 10%
14.7 mL TBS 
SECTION: Immunostaining for TH (Day 1)
10. Rinse 3 times with TBS (10 min each)
SECTION: Immunostaining for TH (Day 1)
11. Blockade with BSA 3% or other blocking solutions 1h (if needed)
SECTION: Immunostaining for TH (Day 1)
12. Primary Antibody (diluted in TBS): overnight at 4°C (or longer period if needed)
TH (1:1000) Pel-Freez, Rogers, AR
SECTION: Immunostaining for TH &amp; Mounting (Day 2)
13. Rinse 3 times with TBS (10 min each)
SECTION: Immunostaining for TH &amp; Mounting (Day 2)
14. Secondary Antibody (diluted in TBS without) 40-45 min Room Temperature (1:400)

SECTION: Immunostaining for TH &amp; Mounting (Day 2)
15. Rinse 2 times with TBS (10 min each) AND Rinse 2 times with TB (10 min each)
SECTION: Immunostaining for TH &amp; Mounting (Day 2)
16. Mounting with Vectashield or PVA DABCO