eamag/protocols-with-steps huggingface dataset:
TITLE: SPARC Segmentation Conversion: Binary mask tracings to MBF XML
AUTHORS: Maci Heal, Susan Tappan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Convert binary mask tracings from Nikon's NIS Elements software into MBF XML format.</div></div>

[STEPS]
?. [Conversion]
Launch Neurolucida 360.
?. [Conversion]
Open binary mask tracings (.tif) into program window.
?. [Conversion]
Select the Outline Objects icon from the Trace ribbon.
?. [Conversion]
Click on a black or white pixel on the binary mask tracings to automatically contour the representative object. Repeat until all objects are contoured.
?. [Conversion]
Save file as XML.
?. [Conversion]
Add subject metadata and select criteria for anatomic term list in the SPARC Vocabulary Services welcome dialog.
?. [Conversion]
Choose the approrpiate vocabulary term from the contour selection toolbar.
TITLE: Collection and shipment of specimen for Bulk RNA-sequencing (Bulk RNA-seq)
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNet Consortium. Here we provide details on specimen collection and shipment to the Robson laboratory at The Jackson Laboratory for Genomic Medicine (JAX-GM) in Farmington, CT for its processing for Bulk RNA-sequencing (Bulk RNA-seq).

[STEPS]
SECTION: Reagents and Materials:
1. 2mL Eppendorf tubes or 5 ml Eppendorf tubes
RNA Later solution
Wet Ice
Tweezers (clean, sterile)
SECTION: Quality Key Points:
2. The tissue specimen should be always kept at 4 degrees Celsius and RNase-free. 
It is crucial to not store the tissue specimen at RT to avoid any cell death, and tissue and/or RNA degradation. 
SECTION: Procedure:
3. Timeline: The daily shipping deadline at Jax BH is 12:00 noon. So, harvest the tissues before and closer to noon.
SECTION: Procedure:
4. Collection/Harvest:
SECTION: Procedure:
4.1. Animal was euthanized via cervical dislocation. 
SECTION: Procedure:
4.2. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest. 
SECTION: Procedure:
4.3. Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
4.4. Heart, pancreas, and kidney was collected and the specimen was transferred into tubes containing ice-cold RNA later (the volume will depend on the size of the specimen, but should cover abundantly the tissue and fill up the tube)
SECTION: Procedure:
4.5. Individually parafilm the Eppendorf tube lids to their tubes before shipping.
SECTION: Procedure:
4.6. Keep at 4 degrees Celsius thereafter, until shipping.
SECTION: Procedure:
5. Shipping:
SECTION: Procedure:
5.1. Place sample tubes in a plastic box (cardboard boxes insulate the samples from the cold ice) or in double Ziplock bags after checking that they are completely sealed.
SECTION: Procedure:
5.2. Ship the sample box on wet ice.
SECTION: Procedure:
5.3. Ship O/N on ice/ice pack, to:
The Jackson Laboratory for Genomic Medicine,
Farmington, CT, 06032
TITLE: Tissue delipidation
AUTHORS: yang1832 , Julia Laskin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This step is to wash lipids off the tissue to prevent their interferences for imaging of proteins.</div></div>

[STEPS]
?. Immerse the tissue section in 70%, 90%, 100% ethanol, 20s each.
?. Immerse in 99.8% chloroform for 25s.
?. Dry in desiccator for 10min.
TITLE: Automated Immunohistochemistry Staining
AUTHORS: Toby J Curless, Hemanth Ramesh Nelvagal, Zane Jaunmuktane

[DESCRIPTION]
The protocols describes the steps for automated immunohistochemistry staining using the Ventana Discovery® XT
Immunostainer.




[STEPS]
SECTION: Preparation
2. Generate tissue sections using standard microtome sectioning protocols.
SECTION: Preparation
1. Heat tissue dry tissue sections for 60 min at 60 °C
SECTION: Preparation
3. Prepare primary and secondary antibody solutions as per manufacturer's protocol - see below for example for AT8.
SECTION: Preparation
4. Primary AT8 antibody (Thermo Fisher Scientific, MN1020) diluted at 1:100 in antibody diluent solution
(Ventana). Secondary anti-mouse antibody (Abcam) diluted at 1:100 in antibody diluent solution (Ventana).
SECTION: Preparation
5.  Perform immunohistochemical staining using the Ventana Discovery® XT Immunostainer – following manufacturers guidelines.

 
SECTION: Preparation
6. Print barcoded slide labels corresponding to the correct protocol on the Ventana machine and stick them to the top of each slide following the slide heating.
SECTION: Processing Slides Using the Ventana Machine
7. Place slides into the Ventana machine and ensure all bulk reagents are sufficiently filled
SECTION: Processing Slides Using the Ventana Machine
8. Begin the staining protocol.
SECTION: Processing Slides Using the Ventana Machine
9. Upon completion of the staining, activate the counterstaining step.
SECTION: Processing Slides Using the Ventana Machine
10. Following counterstaining, remove the slides and wash in soapy water 5x.
SECTION: Processing Slides Using the Ventana Machine
11. Leave slides in running water for 5 min mins.
SECTION: Processing Slides Using the Ventana Machine
12. To deparaffinise sections, place slides in 100 % ethanol for 5 min mins, remove and place in separate 100 % ethanol for 5 min mins. Remove from ethanol and place in xylene for 5 minmins, remove and place in separate xylene for5 minmins

SECTION: Processing Slides Using the Ventana Machine
13. For cover-slipping of slides following deparaffinisation – use the Leica CV5030 automated slide cover-slipping machine.
SECTION: Processing Slides Using the Ventana Machine
14. Stained slides digitised on a NanoZoomer S360 scanner (Hamamatsu) – Brightfield scan profile at x40 magnification.
SECTION: Processing Slides Using the Ventana Machine
15. Upload digitised slides to NZ Connect (1.0.36 (IVD)) slide viewing platform.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A collection of protocols released as part of the IOS EDGE: Functional-genomics tools for Cnidarian-dinoflagellate symbiosis NSF grant (#1645164).</p>
<p> </p>
<p><strong>Lead PIs: </strong></p>
<p>Virginia Weis, Oregon State University</p>
<p>Mauricio Rodriguez-Lanetty, Florida International University</p>
<p>Matthew DeGennaro, Florida International University</p>
<p>Arthur Grossman, Carnegie Institution for Science</p>
<p>John Pringle, Stanford University</p>
<p> </p>
<p><strong>Protocols for getting started w/ the Aiptasia-<em>Symbiodinium</em> Model System:</strong></p>
<p>-General husbandry and culturing of Aiptasia and <em>Symbiodinium </em>in the laboratory</p>
<p>-DNA, RNA, and protein extraction</p>
<p>-Embedding animals for microscopy</p>
<p>-Genotyping</p>
<p> </p>
<p> </p>
<p> </p>

[STEPS]
TITLE: UC Davis - Inflammation pathway
AUTHORS: Fawaz G. Haj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
This test is designated to determine if rodents exhibit inflammatory disorders, through evaluation of the activation state of key proteins such as JNK and p38 MAP kinases. We will examine the inflammatory state of adipose and liver tissues.</div></div>

[STEPS]
?. Unless otherwise requested by the PI or stated in the protocol, mice will be euthanized using cervical dislocation.
?. Collect maximum blood from portal vein and isolate plasma according to standard protocols or as desired by the P.I.
?. Quickly collect tissues designated by the P.I. Each tissue should be divided into three portions, one portion should be snap frozen in liquid nitrogen, one portion should be kept into RNA later solution and the third one should be fixed into the appropriate fixative solution. Please note that the whole procedure of tissue collection should be done within 3 minutes maximum.
?. For western blotting, tissues will be lysed into the appropriate lysis buffer.
?. Total protein expression of MCP1/5, IL-6 and TNF-alpha, activation of JNK and p38 in adipose tissue and/or liver (or any other tissue if requested by the P.I.) will be determined according to the standard Western blotting protocols.
?. Note:
Evaluation of the activation state of other component of inflammation, such as circulating levels of cytokines is also possible upon special request. Extra charges may apply.
Gene expression of pro-inflammatory cytokines is also soable if requested by the P.I. Extra charges may apply.
Evaluation of the activation state of other component of the ER stress and ER stress-associated signaling, particularly the unfolded protein response (such as ERAD proteins, calnexin, calpains, etc...), JNK pathway or ER stress-induced apoptosis is also possible upon special request. Extra charges may apply.
Immunohistochemistry of inflammation markers such as TNFα and MCP1 couldbe performed on fixed tissues if desired by the P.I. Extra charges may apply.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Cycloplegic agents are used routinely during the examination in pediatric patients to investigate the correct refraction without accommodation.</p>
<p>Cycloplegia was performed using the following regimen. Cyclopentolate hydrochloride 1% (Cyplegin 1% ophthalmic solution, Santen Pharmaceutical, Osaka, Japan) was instilled three times at 10-min intervals. The measurements were obtained 60 min after the last instillation. After cycloplegia was established, the lens thickness was routinely determined using the biometer, and the refraction was obtained with the autorefractometer.</p>

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

[DESCRIPTION]
<p>Environmental and housing conditions experienced by laboratory animals exert profound effects on their biology, physiology, and behavior.  These parameters are important and often overlooked sources of potential variation in experiments, and should be reported in peer-reviewed publications in order to promote scientific reproducibility.  To that end, here we provide a summary of the environmental conditions in zebrafish (<em>Danio rerio</em>) at tanks at Laboratório de Neurociências e Comportamento 'Frederico Guilherme Graeff' (LaNeC), located in the Universidade Federal do Sul e Sudeste do Pará, Marabá/PA, Brazil. Included are data on the physico-chemical characteristics of the water, as well health, of zebrafish in separate tanks at LaNeC. </p>
<p>This protocol was based on Lawrence's (https://dx.doi.org/10.17504/protocols.io.mrjc54n), and is forked from that. </p>

[STEPS]
TITLE: Bacterial Transformation - Mix & Go Competent Cells - CHEM 584 
AUTHORS: Ken Christensen

[STEPS]
?. Add  to  plasmid DNA to a  aliqanuot of thawed competent cells (Mix & Go) on ice, mix gently for a few seconds (try to keep the added volume of DNA less than 5% of the total). Place tube on ice for   minutes.
1 µl
5 µl
50 µl
When selecting with Kanamycin, Tetracycline, etc., an outgrowth performed in SOC medium is required for efficient transformation. After the transformation mixture has incubated on ice for at least 5  min,  add  4  volumes of  SOC  (e.g., 400  μl of  SOC  to  100  μl of the transformation mixture) and incubate for 1 hour at 37°C with gentle shaking at 200-300  rpm.
?. Spread    to  of  the mixture onto a  pre-warmed  (37°C)  LB  plate containing Ampicillin (or other selection antibiotic).
50 µl
100 µl
Using chilled LB plates will decrease the transformation efficiency.
?. Incubate overnight at  in the bacterial incubator.
37 °C
You can also incubate on the bench or in a drawer at room temperature for a longer time if this is convenient for scheduling. For example, leaving a plate on the bench for 2-3 days at room temperature usually generates nice sized colonies and you avoid having to come into the lab the next day (e.g., over the weekend).
?. Prewarm culture plates in a  incubator for  .
37 °C
TITLE: Human Knee Meniscus Collection Protocol for scRNA-seq
AUTHORS: molmer, Martin Lotz

[DESCRIPTION]
Meniscus is taken from the medial knee compartment is collected for scRNA-seq processing. The attached image indicates where the meniscal tissue is collected from.

[BEFORE_START]
Knee blocks are shipped on wet ice and received within 48 hours post-mortem from certified tissue banks. The average time from receiving the sample to harvesting cartilage from both knees is about 2 hours. Pictures are taken of knee tissues and macroscopic scoring of the cartilage is completed.

[STEPS]
1. Prepare harvesting area with sterile drapes, tools and gauze. All harvesting is completed within an aseptic environment.

2. Wipe down the knee blocks with 95% ethanol prior to opening the joint capsule.
3. Once the joint capsule is opened, the femur and tibia are disarticulated and menisci are resected.

4. A full-thickness (sagittal) piece of medial meniscus is cut out using a #21 scalpel; the piece measures about 1.60cmX15mmx8mm and weighs around 1g. This is collected into a 50mL tube that is filled with 40mLs of + 1% , then placed in wet ice until the cell isolation protocol for scRNAseq begins.  
 

SECTION: Tissue Collection for MERFISH
9. Procurement of Medial Femoral Cartilage With Subchondral Bone for MERFISH: Using an autoclaved 12” hacksaw with 24TPI blade, cut a 3-5mm slab from the middle region of the medial condyle. 
SECTION: Tissue Collection for MERFISH
0.9. Three pieces of tissue are placed in 12mLs, per region, of   for 24hrs on a 4oC rocker. 

SECTION: Tissue Collection for MERFISH
0.9. Pour off fixation solution and replace with    and store at 4oC until shipment on wet ice or MERFISH protocol begins.
TITLE: Immunofluorescence on paraffin sections
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Immunofluorescence protocol on paraffin-embedded rodent brain sections 

[STEPS]
SECTION: 1. Deparaffinization and hydratation :
1. Put the slides 30 min in the incubator at 60ºC
SECTION: 1. Deparaffinization and hydratation :
2. Wash 3x3 min in xylene
SECTION: 1. Deparaffinization and hydratation :
3. Wash 1x10 min in ethanol 100%
SECTION: 1. Deparaffinization and hydratation :
4. Wash 1x10 min in ethanol 95%
SECTION: 1. Deparaffinization and hydratation :
5. Wash 1x5 min in ethanol 70%
SECTION: 1. Deparaffinization and hydratation :
6. Wash 2x5 min in PBS 1X
 

SECTION: Antigen retrieval
7. Incubate slides in citrate buffer 10mM, pH 6 in a boiling water bath (95ºC) for 20 min
SECTION: Antigen retrieval
8. Let sections cool down for 20 min at room temperature (RT)
SECTION: Washing
9. Wash 3x5 min in PBS 1X-Triton (0.5%)
SECTION: Blocking
10. Put wet paper in a black box for immunohistochemistry
SECTION: Blocking
11. Gently dry and circle sections with the hydrophobic pen ImmEdge Vector H 4000 without touching the tissue 
SECTION: Blocking
12. Add 200 uL/section of blocking solution (5% NGS (in PBS 1X) (200uL/section) +  0.1% Triton (in PBS 1X)) 1h at RT
SECTION: 1ary antibody
13. Gently wipe off the water ofthe slides
SECTION: 1ary antibody
14. Put 200ul/section of 1ary antibody in 2% NGS + 0.1% Triton overnight at 4ºC (cold room)
SECTION: Washing
15. Wash 3x5min in PBS 1X-Triton (0.5%)
SECTION: 2ary antibody
16. Gently wipe off the water ofthe slides
SECTION: 2ary antibody
17. Put 200ul/section of 2ary antibody in 2% NGS + 0.1% Triton + Hoechst/DAPI for 1 h at RT

SECTION: Washing
18. Wash 3x5min in PBS 1X 
SECTION: Mounting
19. Mount sections with fluorescent mounting medium. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles
SECTION: Storage
20. Let dry the slides on the tray in the hood for 5 minutes and store at 4ºC asap
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We describe a chemical-genetic approach for inducible silencing of <em>Caenorhabditis elegans </em>neurons in intact animals, using the histamine-gated chloride channel HisCl1 from <em>Drosophila</em> and exogenous histamine. Administering histamine to freely moving <em>C. elegans</em> that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes. <em>C. elegans</em> does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type <em>C. elegans</em> behavior. </p>

[GUIDELINES]
<p>Gloves are recommended while making or handling histamine-containing liquids. Avoid contact with eyes and skin. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: Colony PCR for screening transgenic yeast 
AUTHORS: Anbarasu  Karthikaichamy

[DESCRIPTION]
Colony PCR protocol for screening transgenic yeast

[BEFORE_START]
Prepare 10 millimolar (mM) sodium hydroxide (NaOH)


[STEPS]
1. Pick single yeast colony from the transformation plate and re-streak to a fresh plate with appropriate selection marker.
  
  

2. Incubate the plate in a 30 °C incubator for 2 days.

3. After 2 days, pick around 8 colonies from the re-streaked plate using the smallest micropipette tip or sterile wooden toothpick.
4. Re-suspend the cells into 20 µL of  10 millimolar (mM) sodium hydroxide (NaOH) by twirling the pipette tip or wooden toothpick inside the NaoH solution.

5. Microwave the PCR tubes at the highest power setting for 5 min.

 



6. Take the PCR tubes out of the microwave oven and briefly spin to pellet down the cells using a bench-top centrifuge.
7. Without disturbing the pellet, carefully aspirate 2 µL out of the tubes and use it as template for PCR.

8. The PCR reaction mix and the primer annealing temperatures can be adjusted depending on the polymerase and the primer pair used.
TITLE: Economic and easy bacterial and yeast colony PCR with 2x premix Gotaq-Green (Promega) or DreamTaq-Green (Thermo Fisher) 
AUTHORS: Darek Abramczyk

[DESCRIPTION]
Slightly modified method Gotaq Promega or DreamTaq (Thermo), economical, quick and ready-to-load. Perfect for yeast genotyping and screening


[STEPS]
1. Bacterial colony PCR 
  
 PCR reactions using ProFlex PCR system, Applied Biosystems. Thermal Cycler
2. Yeast colony PCR (S.cerevisiae, Pichia, Candida)

1.1.  
Reagent NameInitial Conc.Volume (μL)gotaq green 2x2X6.25oligo 1oligo 210 uM 10 uM 0.75 0.75cell suspension in water1ddH2ON/A3.75TOTAL Volume:12.5 uL
  
 Stage NumberNumber of CyclesStep NumberStep NameTemperatureDuration111Initial Denaturation9810min2301Denaturation9810 sec2Annealing50/5530 sec3Extension721min  (depends on the length)311Hold10
 
 

 

1.2. Load on 1-1.2% agarose gel 8-10 µL and visualise under GelDoc or another UV source.



2.1. Alkaline yeast cells lysis treatment. Pick a yeast colony from agar plate and swirl in sterile 20mM NaOH 50-100 µL . 
Optionally, a liquid culture or a agar-plate pick colony resusupend in the sterile water or YPD, take small volume (~20uL) and mix with the same volume of 40mM NaOH.
20mM NaOH-cells resuspension incubate 95 °C 10 min and immediately transfer to ice/4 °C for at least 5 min 


2.2. 
 
 
Reagent NameInitial Conc.Volume (μL)gotaq green 2x2X6.25oligo 1oligo 210 uM 10 uM 0.75 0.75cell suspension in water1ddH2ON/A3.75TOTAL Volume:12.5 uL
   
ABCD Number of CyclesStep NameTemperatureDuration1Initial Denaturation98oC10min30Denaturation98oC10 secAnnealing50-55oC30 sec1min  (depends on the length)72oC1min per 1kb1Hold4-10oChold
 
 
 

 

 
2.3. Load on 1-1.2% agarose gel 8-10 µL and visualise under GelDoc or another UV source.
TITLE: Radiolabeled polyamine uptake in cells
AUTHORS: Marine Houdou, Nathalie Jacobs, Peter Vangheluwe

[DESCRIPTION]
This protocol provides a technique to determine the radiolabeled polyamine uptake capacity in cells, via the acquisition of disintegrations per minute (DPM) using a Liquid Scintillation Counter. 

[GUIDELINES]
Proper guidelines for working with radiolabeled materials should be followed at all times. 

[STEPS]
1. Cells are seeded in 12-well plates, such that 70-80% confluency is reached on the day of the assay. Seed out 2 'treatment' wells, 1 'quick wash' well and 1 'untreated' well per cell line and treatment dose. 
 

2. Remove the culturing medium in 'treatment wells' and add 500 µL of medium, containing the desired concentration of radiolabeled polyamines. Leave regular culturing medium in the 'quick wash' and 'untreated' wells. 

3. Incubate 37 °C 30 min 

4. 4 minutes before reaching the 30-minute mark, perform a Quick-wash step as follows: add 500 µL of medium, containing the desired concentration of radiolabeled polyamines in the 'quick wash' wells.   immediately remove the treatment medium, and wash with 500 µL of DPBS (-/-) containing  50 micromolar (µM) of the respective unlabeled (=cold) polyamine at 4 °C . Continue with 2 more washing steps with 800 µL of DPBS (-/-) 4 °C . 

5. Proceed with washing the other wells ('treatment' and 'untreated'). Remove the treatment medium, and wash with cold 500 µL  of DPBS (-/-) containing  50 micromolar (µM) of the respective cold polyamine at 4 °C . Continue with 2 more washing steps with 800 µL of DPBS (-/-) at 4 °C . 

6. After the last wash, add 200 µL 0.1% SDS-DPBS (-/-) per well to lyse the cells. 

7. Incubate 10 min at Room temperature . 


8. Scrape the cells and pipette the lysates into scintillation vials, that are filled with 7 mL of scintillation fluid (Ecolite (+), MP). Rinse each well with 200 µL  DPBS (-/-) and pipette into the respective scintillation vial. 

9. Mix scintillation vials well prior acquisition of disintegrations per minute (DPM) in the Liquid Scintillation Counter.
TITLE: Culturing Primary Cortical Neurons
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to isolate and culture primary cortical neurons from mouse embryos at E14.5-E15.5.

[STEPS]
SECTION: Day 1
1. Coat culture dishes with 0.05mg/mL Poly-D-lysine overnight at 37oC.
SECTION: Day 2
2. Wash culture dishes with sterile water x2 then leave to dry.
SECTION: Day 2
5. Isolate uterus with embryos and place in a tube of 1X PBS.
SECTION: Day 2
6. In a laminal flow hood, dissect the brains of E14.5-15.5 embryos in Hank's Balanced Salt Solution (HBSS) and isolate each brain in 700uL of HBSS.
SECTION: Day 2
7. Gently pipette up/down x10 to mix cortices then add 20uL trypsin for every embryo and incubate on a rotator at 37oC for 20 minutes.
SECTION: Day 2
8. Add 300uL Solution A per embryo and carefully pipette up/down x5 then centrifuge at 2500g for 5 minutes at 4oC.
Solution A: 2.75mL Neurobasal media (unsupplemented) + 150uL trypsin inhibitor + 100uL DNAse1
SECTION: Day 2
9. Remove supernatant and carefully resuspend pellet in 300uL Solution B per embryo by pipetting up/down x10.
Solution B: 2.55mL Neurobasal media (unsupplemented) + 200uL trypsin inhibitor + 250uL DNAse1
SECTION: Day 2
10. Centrifuge at 2500g for 5 minutes at 4oC then remove supernatant.
SECTION: Day 2
11. Resuspend pellet in 1mL Neurobasal complete per embryo.
SECTION: Day 2
11.1. Optional trypan blue cell exclusion assay: Mix 100uL trypan blue + 100uL 1X PBS + 20uL resuspended cells. Count with haemocytometer.
SECTION: Day 2
12. Plate neurons on pre-coated dishes at desired confluency.
SECTION: Day 2
13. Change neurobasal medium at 4 days in vitro.
SECTION: Day 2
14. Fix neurons at 7 days in vitro by removing media and replacing with 4% paraformaldehyde for 10 minutes at room temperature.
SECTION: Day 2
15. Wash neurons with 1X PBS x2 to remove traces of paraformaldehyde then store in 1X PBS at 4oC.
SECTION: Day 2
3. Sedate pregnant mouse with 120mg/kg Euthanyl and confirm sedation prior to proceeding with the next step.
*The neurons must be plated within approximately 4 hours of sedation.
SECTION: Day 2
4. Soak pregnant mouse with 70% ethanol then dissect open the abdominal cavity.