eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The complexity of ADCs consisting of payloads, and conjugated antibodies and<a href="https://www.creative-biolabs.com/adc/classify-adc-linkers-7.htm" target="_blank" rel="noopener noreferrer"> ADC linkers </a>creates difficulties in developing new pharmacokinetic research methods, understanding the in vivo processes of drugs, and the relationship between therapeutic effects and safety, and thus new organisms. Analytic method research and development are increasingly valued. Recent literature on<a href="https://www.creative-biolabs.com/adc/classify-adc-linkers-7.htm" target="_blank" rel="noopener noreferrer"> ADC characterization</a>, biodistribution, metabolism, and elimination can be found in the importance of research and development of antibody drugs, particularly ADC drugs. Biological analysis of <a href="https://www.creative-biolabs.com/adc/services.htm" target="_blank" rel="noopener noreferrer">ADCs </a>is challenging compared to small molecule drugs and protein therapies because ADCs have a complex molecular structure that has the molecular characteristics of combining small molecule drugs with protein therapies. The ADC runs from the serum or plasma to the site of action or to the target cell. Under the action of the enzyme, the antibody-linker-active drug is cleaved to release the drug and produce a therapeutic effect. For this novel drug action method, the bioanalytical method must take into account the antibody-linker-active drug, and it needs to be specially designed for this complex ADC delivery system to provide more and more complete pharmacokinetic information, but cannot be achieved by traditional biological analysis methods.</p>

[STEPS]
TITLE: GI Transit Assay
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
This test is used to check for possible signs of constipation and alteration in the gut transit 

[STEPS]
1. Fast mice for 6 hours
2. Administer 0.3 mL Carmine red per mouse via oral gavage
3. Monitor the mice every 15 minutes for up to 5 hours for the appearance of the first red fecal pellet 
4. The time from gavage to passage of first red pellet is recorded as total intestinal transit time.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to transform chemically competent <em>Vibrio natriegens</em> cells.</p>
<p>The protocol was described and published by Weinstock et al., 2016</p>

[BEFORE_START]
<p>You need:</p>
<ul>
<li>Chemically competent <em>V. natriegens </em>cells</li>
<li>BHI + V<sub>2</sub>-Salts</li>
</ul>
<p> </p>
<p><strong>10x V<sub>2</sub>-Salts:</strong></p>
<table style="height: 41px;" width="599">
<tbody>
<tr>
<td style="width: 142px;">Chemical</td>
<td style="width: 143px;">Concentration</td>
<td style="width: 143px;">Molecular weight</td>
<td style="width: 143px;">Amount for 1L</td>
</tr>
<tr>
<td style="width: 142px;">NaCl</td>
<td style="width: 143px;">204mM</td>
<td style="width: 143px;"> 58.44g/mol</td>
<td style="width: 143px;">11.92176g</td>
</tr>
<tr>
<td style="width: 142px;">KCl</td>
<td style="width: 143px;">4.2mM</td>
<td style="width: 143px;">74.55g/mol </td>
<td style="width: 143px;">0.31311g</td>
</tr>
<tr>
<td style="width: 142px;">MgCl<sub>2</sub></td>
<td style="width: 143px;">23.14mM</td>
<td style="width: 143px;">203.3g/mol </td>
<td style="width: 143px;">4.704362g</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>BHI:</strong></p>
<p>For 1L medium 37g Brain heart infusion buillon</p>

[STEPS]
?. 
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TITLE: Vandy – Post Clamp Anesthesia
AUTHORS: Louise Lantier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">After the last sample is taken for the clamp, the mouse is anesthetized in order to harvest tissues. It is very important that the mouse remain alive during tissue harvesting for future signaling or metabolite analysis. The mouse is therefore given a sub-lethal dose of pentobarbital to induce anesthesia (~70mg/kg pi or 35mg/kg iv). </div></div>

[STEPS]
?. Draw up the desired volume of diluted Pentobarb (5mg/ml in saline) in a blunted syringe.     • To give 35mg/kg, multiply body weight (grams ) by 7, to get the volume to inject in uL.     • Pentobarb (uL) = BW (g) * 7
?. Steadily inject the pentobarbital in the jugular line of the catheterized mouse.
?. If the mouse is not completely anesthetized when taking the tissues, inject more by 50uL increments.
TITLE: Optogenetic experiments with iLID system
AUTHORS: Andrés Guillén-Samander, Pietro De Camilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details experiments with the iLID optogenetic system as performed to acutely recruit Miro to mitochondria in </span><a href="https://doi.org/10.1083/jcb.202010004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1083/jcb.202010004</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Optogenetic experiments with iLID system]
Plate the COS-7 cells for imaging and transfect each MatTek dish (see Cell Culture, Transfection and Imaging Protocol) with the plasmids containing the bait (construct encoding iLID) and the prey (construct encoding SspB peptide).
As an example, in the case of Guillén-Samander A, Leonzino M et al 2021 (https://doi.org/10.1083/jcb.202010004), the plasmids and amounts transfected were:   mCh-Miro1∆TM-SspB  Venus-Mito-iLID   VPS13D^HaloSuggestion: When dealing with an optogenetic pair of constructs encoding a membrane anchored protein and a cytosolic one, we recommend transfecting them in a ratio of 2:1 for membrane: cytosolic proteins.
?. [Optogenetic experiments with iLID system]
After adding the transfection mix, incubate the dishes at  in the dark for -.
37 °C
?. [Optogenetic experiments with iLID system]
Just before imaging, remove the growth medium and replace with Live Cell Imaging solution (Life technologies).
?. [Optogenetic experiments with iLID system]
Image the cells at  and 5% CO2.
37 °C
Warning: Remember to keep the cells in the dark while handling them and as much as possible while changing the medium. Exposure to light could preactivate the optogenetic system which could affect the interpretation of results.
?. [Optogenetic experiments with iLID system]
Carry out imaging in an Andor Dragonfly system equipped with a PlanApo objective (63X, 1.4NA, Oil) and a Zyla sCMOS camera.
?. [Optogenetic experiments with iLID system]
Identify the cells to be imaged by scanning the dish looking for red fluorescence in the 564nm channel.
Remember that looking for green fluorescence would preactivate your sample.
?. [Optogenetic experiments with iLID system]
Image the cells at a rate of 0.5Hz. Acquire the 5 frames before the activation, and achieve the activation with a single 200ms pulse of the 488nm laser. Acquire the 90 frames after activation.
?. [Optogenetic experiments with iLID system]
Carry out imaging in a Nikon Ti-E inverted microscope equipped with an Improvision UltraVIEW VoX system (Perkin Elmer) and a PlanApo objective (63X, 1.4NA, Oil) and controlled by Volocity software (Quorum Technologies).
?. [Optogenetic experiments with iLID system]
Identify the cells to be imaged by scanning the dish looking for red fluorescence in the 564nm channel.
Remember that looking for green fluorescence would preactivate your sample.
?. [Optogenetic experiments with iLID system]
Image the cells at a rate of 0.5Hz. Acquire 5 frames before the activation, and achieve the activation by delivering a single 488nm light pulse in a 5 μm2 area using a built-in photo-perturbation unit.
TITLE: BG11 hypersaline medium
AUTHORS: Tanja Bosak

[STEPS]
?. Add the following to a beaker and dissolve using a stir bar on a magnetic stir plate.  ABC1BG11 hypersaline mediumamountunit2initial Milli-Q water968ml3NaCl49.8g 4NaNO31.5g5Na2CO30.02g6KCl1.3867g7MgCl2*6H2O8.569g8MgSO4*7H2O6.4998g9CaCl2*2H2O2.042g10Stock A10ml11Stock B10ml12Stock C10ml13Stock 5 (trace metals)1ml14Vitamins1ml
ABC1BG11 hypersaline mediumamountunit2initial Milli-Q water968ml3NaCl49.8g 4NaNO31.5g5Na2CO30.02g6KCl1.3867g7MgCl2*6H2O8.569g8MgSO4*7H2O6.4998g9CaCl2*2H2O2.042g10Stock A10ml11Stock B10ml12Stock C10ml13Stock 5 (trace metals)1ml14Vitamins1ml
?. Filter sterilize near Bunsen burner or in sterile biohood.
?. STOCK SOLUTIONS  AB1Stock AConcentration (g/L)2Na2MG EDTA0.13Ferric ammonium citrate0.64Citric acid*1H2O0.65CaCl2*2H2O3.66Add up to 1L of dH20   AB1Stock BConcentration (g/L)2MgSO4*7H2O7.53Add up to 1L of dH20   AB1Stock CConcentration (g/L)2K2HPO4*3H2O43Add up to 1L of dH20   AB1Stock 5Concentration (g/L)2H3BO32.863MnCl2*4H2O1.814ZnSO4*7H2O0.2225CuSO4*5H2O0.0796CoCl2*6H2O0.057NaMoO4*2H2O0.3918Add up to 1L of dH20
AB1Stock AConcentration (g/L)2Na2MG EDTA0.13Ferric ammonium citrate0.64Citric acid*1H2O0.65CaCl2*2H2O3.66Add up to 1L of dH20
AB1Stock BConcentration (g/L)2MgSO4*7H2O7.53Add up to 1L of dH20
AB1Stock CConcentration (g/L)2K2HPO4*3H2O43Add up to 1L of dH20
AB1Stock 5Concentration (g/L)2H3BO32.863MnCl2*4H2O1.814ZnSO4*7H2O0.2225CuSO4*5H2O0.0796CoCl2*6H2O0.057NaMoO4*2H2O0.3918Add up to 1L of dH20
?. Add Milli-Q water to bring total volume to 1 L.
?. Adjust pH to ~7 - 7.4
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the quick protocol for the Q5&reg; Site-Directed Mutagenesis Kit (E0554)

[BEFORE_START]
Primers should be designed with 5&acute; ends annealing back-to-back. We recommend using the NEB online design software,&nbsp;<a href="http://nebasechanger.neb.com/" target="_blank">NEBaseChanger&trade;</a>.

[STEPS]
?. 
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TITLE: Histology: Analysis Protocol
AUTHORS: Sasha Burwell

[DESCRIPTION]
This protocol details the analysis of histology images, including usage of Matlab codes to draw regions of interest (ROIs) and  ilastik software to perform pixel classification.

[STEPS]
SECTION: Analysis Protocol
1. Go to the folder with all of the .vsi images from one brain.
SECTION: Analysis Protocol
2. Use the 
 
 Matlab code to manually draw regions of interest (ROIs) of the left VTA and right VTA per section.
SECTION: Analysis Protocol
2.1. Change the channels and intensities (top right table) as needed to visualize your signal.
SECTION: Analysis Protocol
2.2. Make sure the tissue is oriented correctly (left hemisphere on the left) – use the “VS200” button to rotate, and the “FlipLR” button to flip horizontally, as necessary.
SECTION: Analysis Protocol
2.3. Click the “PP” button to draw two polygons.

Left VTA first, then right VTA.
After both polygons are drawn, you can move points on the polygon to edit them.
SECTION: Analysis Protocol
3. Repeat for each section with the VTA.
SECTION: Analysis Protocol
4. These drawn ROIs automatically save in a 
 
 structure, but you can click “SaveBkp” to create a backup if there are any issues.
SECTION: Analysis Protocol
5. For pixel intensity calculation:

Run 
 
 to calculate the fluorescence of the specified channels in your drawn ROIs.

Then run 
 
 to extract quantifications from the calculated analysis.
SECTION: Analysis Protocol
6. For cell counting:
SECTION: Analysis Protocol
7. Perform Object Classification:
SECTION: Analysis Protocol
6.2. Run ilastik software (https://www.ilastik.org/documentation/).
SECTION: Analysis Protocol
6.3. Perform Pixel Classification:

Load raw data (the previously created jpg images).
Feature selection:

 
Training: train the algorithm to detect a cell versus not-cell (background tissue) by manually labeling cell and background in various sections.
Once you are happy with the segmentation performed by the classifier, export all predictions.
SECTION: Analysis Protocol
6.1. Save the ROIs as jpg images by clicking “Export” in the 
 
 code GUI, making sure the contrast allows visualization of cells and color of cells and only channels of interest are turned on.
SECTION: Analysis Protocol
7.1. Load raw data (jpg images) and prediction maps (the predictions from pixel classification).
SECTION: Analysis Protocol
7.2. Use standard Threshold and Size Filter settings.

 

SECTION: Analysis Protocol
7.3. Click “Select Features”.
SECTION: Analysis Protocol
7.4. In Object Classification, create 3 label classes: 

red only, 
green only, and 
both.
SECTION: Analysis Protocol
7.5. Train the algorithm to classify these 3 classes by labeling different cells as having red only (ex, only dTomato), green only (ex, only TH or GCaMP), or both (yellow, both proteins).
SECTION: Analysis Protocol
7.6. Once you are happy with the predictions from the classifier, export the Object Identities as .csv files.
SECTION: Analysis Protocol
8. Use these .csv files to count how many cells were identified in each label class across all the sections.
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Concentration of the O marina cultures
?. 
SECTION: Linearization of the DNA that will be transformed into O marina
?. 
SECTION: Preparation of the reagents for the particle bombardment
?. 
SECTION: Preparation of the gold particles
?. 
SECTION: Preparation of the cultures on the (agarose) plates
?. 
SECTION: Coating the gold particles with the DNA to transfect
?. 
SECTION: Bombardment of the cells
?.
TITLE: Peroxidase/DAB protocol
AUTHORS: Christiana.bjorkli  

[DESCRIPTION]
Peroxidase/DAB protocol for chromogen labelling 

[STEPS]
1. Leave sections in 0.125 Molarity (M) phosphate buffer (PB) at 60 °C 

2. Rinse sections 2 x 10min in PB
3. Rinse sections 1 x 10 min in 0,5% TBS-Tx
3.1. Tris: 3,03 gram/500 mlH2O
NaCl: 4,48 gram/500 mlH2O
Triton-X-100: 2,5 ml/500 mlH2O

Use HCl to adjust the pH 8. Store in refrigerator for up to one week.

1.1. Dilute 0,4M PB. Store in refrigerator for up to 1 week.

100 ml: 31,25 ml0.4 Molarity (M) PB + 68,75 ml H2O

4. Incubate with 10% goat serum in TBS-Tx
5. Incubate with primary antibody (AT8; MAP2) 1:1000 in TBS-Tx with 5% goat serum,overnight in refrigerator 4 °C

6. Rinse 3 x 10 min in TBS-Tx
 

7. Incubate with secondary antibody, biotinylated goat anti-mouse, 1:500 in TBS-Tx with 5% goat serum, in room temperature
 

8. Rinse sections 3 x 10 min in TBS-Tx
9. Incubate with ABC in room temperature
6.1. DAB
Dissolve 1 tablet (10 mg) in 15 ml Tris-HCl by leaving it on a stirrer with heat (max 50 °C) for about 2 hours. Add 12 µL H2O2 just before use and filtrate.

For dissolving the DAB use a brain cup (or any other cup you can throw away)
For filtrating the DAB use a disposable syringe with filter.

 

7.1. From the ABC-kit, put 1 drop of solution A and 1 drop of solution B in 5 mL TBS-Tx. Mix well and leave on the bench for 30 min before use.
10. Rinse 3 x 10 min in TBS-Tx
11. Rinse 2 x 5 min in Tris-HCl
12. Incubate with DAB
13. Rinse 2 x 5 min in Tris-HCl
14. Mount sections in Tris-HCl on Superfrost slides and let them dry
15. Coverslip with Xylene and Entellan
TITLE: Criteria for performing total knee arthroplasty without postoperative intensive care monitoring
AUTHORS: Fabricio Loures, João Henrique Reis, Marcelo de Oliveira Campos, Guilherme Morgado Runco, Luísa Doim Vieira Fonseca, Luciana Ferreira Xavier, Nelson Hiroyuki Miyabe Ooka, Liszt Palmeira de Oliveira

[DESCRIPTION]
After reviewing the medical literature, we defined objective criteria to indicate intensive care monitoring after total knee arthroplasty. These criteria were adopted in January 2020 and since then none of the 80 patients who underwent total knee arthroplasty at the Pedro Ernesto University Hospital and who met the criteria required intensive care monitoring.

[BEFORE_START]
We have defined these criteria based on medical literature and we are validating this protocol by observing the outcome.

[GUIDELINES]
To perform total knee arthroplasty without postoperative intensive care monitoring, it is necessary for the patient to fulfill the eight criteria defined below:

Less than 75 years old
American Society Anesthesiology (ASA) Classification I or II
Non-smoker
No history of ischemic disease (Acute Myocardial Infarction and/or Cerebrovascular disease)
Primary Arthroplasty (CR or PS)
Body Mass Index (BMI) less than 35 kg/m²
Creatinine clearance higher than 60 ml/min
Hemoglobin greater than 12g/dL

[STEPS]
TITLE: CTAB Extraction Protocol for Sediment
AUTHORS: Cameron R. Turner

[DESCRIPTION]
Successfully used by Turner et al., 2015 to detect bigheaded Asian carp surface sedimentary DNA from experimental ponds and natural rivers

https://www.sciencedirect.com/science/article/pii/S000632071400442X

[STEPS]
SECTION: Sample preparation
1. THAW the CTAB-preserved sediment sample in the fridge for no more than 1440 min

DECONTAMINATE the exterior of the sample tube with 10 % bleach solution and rinse with reverse osmosis water
SECTION: Sample preparation
2. VORTEX at max speed for 30 s

INCUBATE at 60 °C for 10 min
SECTION: Chloroform extraction
3. ADD 15 mL of Sevag (Chloroform/Isoamyl alcohol 24:1)
 

SECTION: Chloroform extraction
4. VORTEX the sediment/CTAB/Sevag mixture briefly

SHAKE at low speed (Vortexer setting 4) for 5 min
SECTION: Chloroform extraction
5. CENTRIFUGE at 3220 x g for 15 min at Room temperature

CAREFULLY transfer the supernatant to a new 50 mL tube
SECTION: Ethanol precipitation
6. ADD an equal volume of ice-cold Isopropanol to the supernatant

ADD 1⁄2 volume of 5M NaCl to the supernatant

INCUBATE at -20 °C for 60 min (or overnight if more convenient)
SECTION: Ethanol precipitation
7. CENTRIFUGE at 3220 x g for 15 min at Room temperature 

CAREFULLY pour off the supernatant
SECTION: Ethanol precipitation
8. ADD 2 mL of 70% EtOH, washing down the inner walls of the tube

CENTRIFUGE at 3220 x g for 2 min at Room temperature

POUR off EtOH and allow the DNA pellet to air dry completely (a 45 °C incubator can be used to evaporate stubborn droplets)

SECTION: DNA resuspension
9. RESUSPEND the pellet in 1000 µL of LoTE buffer. Heat briefly at 45 °C and swirl gently to mix and resuspend. Once fully resuspended, briefly centrifuge to collect all liquid in the bottom of 50-mL tube.
SECTION: DNA resuspension
10. Transfer all liquid to a 1.5-μL low-bind microcentrifuge tube
SECTION: DNA resuspension
11. Use 200 µL in OneStepTM Inhibitor Removal Kit (Zymo Research, Irvine, CA)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span class="cit-title">This is a collection of protocols from:<br /></span><span class="cit-auth cit-auth-type-author">Linlin Yin</span><span class="cit-sep cit-sep-separator">, et al.</span><span class="cit-auth cit-auth-type-author"> (2015) <a href="http://www.genetics.org/content/200/2/431.long" target="_blank">Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs</a>. </span>Genetics <span class="cit-vol">200<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">431</span><span class="cit-sep">-</span><span class="cit-last-page">441</span></span><span class="cit-elocation">; </span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.115.176917<br /><br />Please see the<a href="http://www.genetics.org/content/200/2/431.long" target="_blank"> full manuscript</a> for additional details.</span>

[STEPS]
TITLE: Creed / Drago Check List
AUTHORS: Kausalia Vijayaragavan

[DESCRIPTION]
Important to check after any Ionpath servicing. 
The in house preset SIO and Sample Bias values are different from IonPath's default values. 
The values were determined empirically (Hadeesha Piyadasa et.al.) to optimise organic (Noodle) noise and true signal values.


[BEFORE_START]
Important to check after any Ionpath servicing

[GUIDELINES]
Make sure the SIO, Sample Bias and DEM Gain Values are set to Our Specs

[STEPS]
SECTION: Check on SIO Voltage Settings
1. Go to HV Control Tab
SECTION: Check on SIO Voltage Settings
1.1. Check on Voltage of Secondary Ion Optics (SIO) 
 
 
Make sure Lens1 voltage is at or to close 500V. Do not worry about the negative value. If it is at 1000V please contact Mike Angelo. 1000V is the default voltage setting for IonPath which we do not use.

SECTION: Check on Sample Bias and DEM Settings
2. Go to Service UI Tab


SECTION: Check on Sample Bias and DEM Settings
2.1.  
 
Scroll down to bottom of the page

SECTION: Check on Sample Bias and DEM Settings
2.2.  
Select the Setting.ui option 

SECTION: Check on Sample Bias and DEM Settings
2.3. Checks on Sample Bias Value 
Make sure the Sample Bias is set at 66. If not change and hit Submit button.

SECTION: Check on Sample Bias and DEM Settings
2.4. Checks on DEM Gain Value   
 
Scroll down to bottom of the page. Make sure the DEM Gain is set at 0.15. If not change and hit Submit button.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes  a method to perform simultaneous extraction of RNA (using the Qiagen's miRNeasy Mini Kit), genomic DNA and protein from canine mast cell tumour or canine skin biopsies (in the form of 3mm cubes) preserved in RNA-Later.</p>

[BEFORE_START]
<p>You will need to prepare the following solutions for this procedure:</p>
<p> </p>
<p><strong><u>Genomic DNA isolation</u></strong> (prepare using nuclease-free water, and 0.2µm filter-sterilise after preparation):</p>
<ul>
<li>75% (v/v) Ethanol</li>
<li>0.1 M sodium citrate.2H<sub>2</sub>O in 10% (v/v) ethanol (sodium citrate solution)</li>
<li>8 mM NaOH (pH is usually around 9)</li>
<li>0.1 M HEPES </li>
<li>100 mM EDTA</li>
</ul>
<p> </p>
<p><strong><u>Protein isolation</u></strong></p>
<ul>
<li>0.3M Guanidine-hydrochloride in 95% (V/V) ethanol (guanidine-ethanol solution) </li>
<li>
<p>141.6mM  β-mercaptoethanol in 10M urea (urea/ β-mercaptoethanol solution).</p>
<p>Dissolve 0.6g of urea in 8mL of ‘ultrapure water’ by stirring. Heat gently to dissolve completely and adjust the volume to 10mL with ultrapure water. Add 1µl of 14.3M β-mercaptoethanol to 100µl of 10M urea shortly prior to use.</p>
</li>
</ul>

[STEPS]
?. 
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TITLE: seqFISH Tissue Preservation
AUTHORS: Long Cai, Nina Dar, Yiing Lin, Shin Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tissue Preparation for seqFISH</div></div>

[STEPS]
?. [Fixation]
Clean surface with RNAzap.  Make the following solutions.1L PBS 1x, RNAse-free1.	take 1 L RNAse-free water bottle2.	use serologic pipet and draw up 100 mL water into another container3.	add 100 mL of 10x RNase-free PBS4.	mix thoroughly50% sucrose1.	tare 1 L RNase-free disposable container2.	take 500 g RNAse-free sucrose container3.	add RNAse-free water to sucrose and transfer to 1 L container4.	continue to add RNAse-free water until mass to 1 kg5.	mix thoroughly, may need agitation on platform at RT for some time6.	store at 4⁰CNOTE: for fixation, minimum PFA to tissue volume is 20:1, ideal closer to 50:1
?. [Sucrose gradient]
Place the tissue in 10% Sucrose at RT and wait for it to sink in 50 mL conical, add 10 mL of sucrose wait for sample to sink to bottom – minimum incubation time of 30 min remove supernatant for next bath.
?. [Sucrose gradient]
Place the tissue in 30% sucrose in 4⁰C – ready for shipping. Make sure there is enough 30% sucrose solution in container e.g. if in 50 mL conicals, have 50 mL of 30% sucrose.
?. [Sucrose gradient]
Place the tissue in 20% Sucrose at RT and wait for it to sink.
?. [Fixation]
Wash three times with equal volume (30mL) of 1x PBS to remove PFA leave a bit of extra liquid in the bottom between each wash.
?. [Fixation]
Prepare 4% PFA in 1x PBS, 120 mL total for 4 tissues 30 mL each in 50 mL conicals.   AB1prepare 3x conicals for 4 tissues216% PFA10 ml310x PBS4    ml4RNAse free water26 ml5total40  ml
AB1prepare 3x conicals for 4 tissues216% PFA10 ml310x PBS4    ml4RNAse free water26 ml5total40  ml
?. [Fixation]
Place each piece in a 50 ml tube (or smaller if you can cut to smaller pieces) in 4% PFA leave at RT for 3 hrs.** the sample should be completely covered.
?. [Fixation]
Cut freshly dissected tissue to small pieces for heart, try cutting slab, 2 cm x 1 cm x 0.4 cm thickness: 2 cm dimension from epicardium to endocardium.