eamag/protocols-with-steps huggingface dataset:
TITLE: NADH Oxidase Activity
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Describes assay to quantitate NADH Oxidase activity from tissues. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biky4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bikz4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bik24kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bik34kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bik44kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bik54kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bik64kcn.jpg" /></div></div>

[STEPS]
?. [Sample Preparation:]
Turn on Multiskan, set temp to 37ºC and set up plate layout.
?. [Sample Preparation:]
Sonicate tissue on ice in 20mM PB pH 7.4 with PMSF inhibitor or thaw prepared samples on ice.
?. [Sample Preparation:]
Remove 25µL for protein analysis.
?. [Sample Preparation:]
Prepare NADH, enough for whole plate.
?. [Sample Preparation:]
Dilute samples 1:5 with de-ionized H²O.
?. [Sample Preparation:]
Using a clear plate: Add 50µL sample to wells and 50µL diluted sample to wells in duplicate.
?. [Sample Preparation:]
Add 50µL buffer to 3 wells for blanks for positive control.
?. [Sample Preparation:]
Add 100µL Tris-Mes to each sample and blanks.
?. [Sample Preparation:]
For negative control add 200µL Tris-Mes to 3 wells.
?. [Sample Preparation:]
Place plate in Multiskan and add 50µL 600µM NADH to the sample and positive blanks. Do not add NADH to the 3 negative control wells.
?. [Sample Preparation:]
Press start and read at 340nm for 10 minutes @ 1 minute intervals.
?. [Sample Preparation:]
Save raw data as an Excel file into the NADHx data folder. Use the naming convention NAXXXX.xls, where XXXX is the date in mmdd format.
TITLE: Preparing hPSC-derived neurons for single-cell RNA sequencing
AUTHORS: Cortina Chen, Florian T Merkle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about Preparing hPSC-derived neurons for single-cell RNA sequencing.</div></div>

[STEPS]
?. Prepare dissociation solution and wash buffers.
?. Wash cells 2x with DPBS.
?. Add dissociation solution ( for 6 well plate,  for 24 well plate).
1 mL
250 µl
?. Incubate at  for up to  until cells are ready to detach easily (test by pipetting dissociation solution onto cells with P1000, check every ).
37 °C
?. Detach in  and break up by gently pipetting up and down within the well until there are no visible clumps (triturate for ~20x).
[Wash Buffer 1]
?. Pass cells through a 40 μm cell strainer and transfer to a Lobind Eppendorf.
?. Pellet at .
Centrifuge: 200 34, 3 min
?. Aspirate media and wash pellet 2x with  (spin at ).
[Wash Buffer 2]
Centrifuge: 200 34, 3 min
?. Pass cells through 40 μm cell strainer into Lobind Eppendorf.
?. Dilute 1:20 in  and count using Trypan blue and Countess (take average of 3x counts).
[Wash Buffer 2]
Note: If using NucleoCounter, ensure that there is no more than 3% aggregation. If >3%, pass through cell strainer again.
?. Dilute to 4.26 x105 cells per mL and take  to CRUK for 10X library prep (47 μL at this density equates to 20,000 cells).
[cell suspension]
on ice
NotesTo target 10,000 cells -> 47 μL of 2.13 x 105 cells per mLFor Novaseq V3, up to 25,000 cells can be tolerated, but this is not recommended as there will be excess doublets
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is part of the manuscript: <a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a></p>
<p> </p>
<p>We acknowledge the help of the Won-Jae Lee laboratory and the Leulier laboratory for assistance in developing these protocols.</p>

[BEFORE_START]
<p>All bacterial media and bacterial manipulations are performed in a laminar flow hood.</p>

[GUIDELINES]
<p>The following bacterial species and strains (kindly provided by François Leulier, IGFL, France and Won-Jae Lee, SNU, South Korea) were used in this study: <em>Lactobacillus</em> <em>plantarum</em><sup>WJL</sup> [1], <em>Lactobacillus</em> <em>brevis</em><sup>EW </sup>[1], <em>Acetobacter</em> <em>pomorum </em>[1]<em>,</em> <em>Commensalibacter</em> <em>intestini<sup>A911T</sup></em> [1] and <em>Enterococcus</em> <em>faecalis </em>[2].</p>
<p> </p>
<p>All bacterial media and bacterial manipulations are performed in a laminar flow hood.</p>
<p> </p>
<p> </p>
<p>Solid media are prepared as follows:</p>
<ul>
<li>MRS - Mix 51 g/l of MRS (Sigma-Aldrich, #69966), 4 ml/l of Tween 20 (Sigma-Aldrich, #P9416) and 15 g/l agar (Difco, #214530) with milliQ filtered water. Autoclave the medium for 15 minutes at 121ºC and pour into Petri dishes.</li>
</ul>
<ul>
<li>Mannitol medium - Mix 3 g/l Bacto peptone (Difco, #0118-17), 5 g/l yeast extract (Difco, #212750), 25 g/l D-Mannitol (Sigma-Aldrich, #M1902) and 15 g/l agar (Difco, # 214530) with milliQ filtered water. Autoclave the medium for 15 minutes at 121ºC and pour into Petri dishes.</li>
</ul>
<ul>
<li>LB - Mix 35 g/l of LB Broth with Agar (lennox) (Sigma Aldrich #L2897) with milliQ filtered water. Autoclave the medium for 15 minutes at 121ºC and pour into Petri dishes.</li>
</ul>
<p> </p>
<p>Liquid media are prepared as follows:</p>
<ul>
<li>MRS - Mix 51 g/l of MRS (Sigma-Aldrich, #69966) and 4 ml/l of Tween 20 (Sigma-Aldrich, #P9416) with milliQ filtered water. Autoclave the medium for 15 minutes at 121ºC.</li>
</ul>
<ul>
<li>Mannitol medium - Mix 3 g/l Bacto peptone (Difco, #0118-17), 5 g/l yeast extract (Difco, #212750), and 25 g/l D-Mannitol (Sigma-Aldrich, #M1902) with milliQ filtered water. Autoclave the medium for 15 minutes at 121ºC.</li>
</ul>
<ul>
<li>LB - Mix 20 g/l of LB Broth (Sigma-Aldrich #L3022) with milliQ filtered water. Autoclave the medium for 15 minutes at 121ºC.</li>
</ul>
<p> </p>
<p><strong>References</strong></p>
<p><br />1. Ryu J-H, Kim S-H, Lee H-Y, Bai JY, Nam Y-D, Bae J-W, et al. Innate Immune Homeostasis by the Homeobox Gene Caudal and Commensal-Gut Mutualism in Drosophila. Science. 2008;319: 777–782. doi:10.1126/science.1149357</p>
<p><br />2. Cox CR, Gilmore MS. Native Microbial Colonization of Drosophila melanogaster and Its Use as a Model of Enterococcus faecalis Pathogenesis. Infect Immun. 2007;75: 1565–1576. doi:10.1128/IAI.01496-06</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B
AUTHORS: Xuefeng Ren

[DESCRIPTION]
This protocol details generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B.

[STEPS]
SECTION: Procedures
1. Transform each plasmid into Stbl3 competent cells for the propagation. Next day, pick up one  colony to inoculate   culture in LB medium with ampicillin.

SECTION: Procedures
2. Midi prep the cultures to purify plasmids (Qiagen).
SECTION: Procedures
3. Plate 5 x 106 HEK 293T cells on a 10 cm plate in DMEM medium.
SECTION: Procedures
4. HEK293T transfection:
SECTION: Procedures
4.1. Add retroviral packaging plasmids (pVSV-G, pBS-CMV-gagpol) and pMRX-IP-HaloTag7-LC3, 5 µg each in 1.5 mL warm Opti-MEM medium.

SECTION: Procedures
4.2. Add 45 µL of TransIT-LT1 transfection reagent (Mirus) and swirl.

SECTION: Procedures
4.3. Incubate at Room temperature for 15 min.
SECTION: Procedures
4.4. Add 1.5 mL dropwise into 10 cm HEK293T plate.

SECTION: Procedures
4.5. At 72 hours post-transfection, collect retroviral supernatant into a falcon tube.
SECTION: Procedures
5. Concentrate retroviral supernatant to 1 mL using Lenti-X concentrator (Takara) with the manufacturer instruction.

SECTION: Procedures
6. Retroviral transduction:
SECTION: Procedures
6.1. Plate 1 x 105 ATG3 KO HeLa cells into 12-well plate one day before.
SECTION: Procedures
6.2. Next day, titrate 100 µL, 200 µL, 400 µL of concentrated retroviral solution with 8 µg/mL Polybrene (Sigma) into target HeLa cells.

SECTION: Procedures
6.3. At 24 hours post-transduction, remove retroviral supernatant and replace with fresh DMEM  complete medium with 2 µg/mL puromycin (GoldBio).

SECTION: Procedures
6.4. Perform Puromycin selection for two weeks. Confirm Halo-LC3B positive cells by FACS sorting.
TITLE: Cylinder behavioral test
AUTHORS: miquel.vila

[DESCRIPTION]
Cylinder behavioral testfor left and right forepaw use

[BEFORE_START]
All behavioral tests have to be performed during the light cycle by an investigator blinded to the experimental groups.

[GUIDELINES]
Important: Rats that present an asymmetric usage of the right-left forepaws at basal examination need to be excluded from the experiment. 

[STEPS]
1. First allow rats to habituate to the experimental room for at least 1 h before each test.
2. Put rats in a glass cylinder and the total number of left and right forepaw touches performed within 5 min was counted.
3. Clean behavioral equipment with 70% ethanol after each test session to avoid olfactory cues.
TITLE: Food Security And Self Sufficiency Program In Mo’orea
AUTHORS: Nohea Rodriguez

[DESCRIPTION]
This research aims to investigate the impact of food dependency on local livelihoods in Mo’orea in terms of nutrition and food accessibility. Through a culturally sensitive and community-based diet program (The Mo'orea Diet Program), we envision a transition from a Westernized diet to a Tahitian diet. It will also explore opportunities for promoting local self-sufficiency through policies that encourage and enable the increased use of traditional Tahitian foods. This project is crucial for understanding the socio-economic dynamics of food dependency in Mo'orea and identifying strategies for enhancing local resilience and sustainability. By promoting the use of local Tahitian foods, this research has the potential to significantly improve local livelihoods, reduce environmental impact, and foster community resilience in the face of possible global disruptions.

[STEPS]
SECTION: The Mo&#x27;orea Diet Program
1. Interview communities throughout Mo’orea for insight on food consumption patterns, barriers to accessing local foods, and perceptions of policy interventions.
SECTION: The Mo&#x27;orea Diet Program
2. Identify key Tahitian foods and dietary practices that can be incorporated into current diets. This will form the "Mo'orea Diet".
SECTION: The Mo&#x27;orea Diet Program
1.1. Target local residents, farmers, and community elders.
SECTION: The Mo&#x27;orea Diet Program
2.1. Only use foods eaten by Tahitians prior to Western influence. Focus research on culturally significant foods and connect them to traditional Tahitian beliefs. Present them as cultural teaching tools to participants.
SECTION: The Mo&#x27;orea Diet Program
3. Find 20 participants from the Mo’orea community. Participants will be given a non-calorie-restricted diet where they can eat within the diet until satiety.
SECTION: The Mo&#x27;orea Diet Program
3.1. The Mo'orea Diet will be implemented with community partners, and we will provide educational resources and support throughout the program.
SECTION: The Mo&#x27;orea Diet Program
4. Record participants’ baseline health status before they start the diet. This will include any history of non-communicable diseases (NCDs), glucose levels, cholesterol levels, blood pressure, etc.
SECTION: The Mo&#x27;orea Diet Program
5. Throughout a 21-day period, monitor participants’ dietary intakes and health outcomes. 
SECTION: The Mo&#x27;orea Diet Program
6. After the 21 days, conduct the same health assessment (from Step 4) from before the program, and compare both findings. 
SECTION: The Mo&#x27;orea Diet Program
7. After five years, monitor any changes in health outcomes and participant well-being after the program.
SECTION: The Mo&#x27;orea Diet Program
8. Construct Community Engagement Plan based on research findings.
TITLE: 10x Protocols: Visium v2 CytAssist FFPE Deparaffinization, H&amp;E staining, and Imaging-- University of Minnesota TMCs (CG000520 Rev B)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression v2 chemistry on FPPE samples with the CytAssist component.

Protocol ID# (CG) and Revision used: 
    10x protocol CG000520, Revision B -- Deparaffinization, H&E staining, and Imaging prior to Library construction with CytAssist.

Note: These protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. Please review the 10x Genomics support website for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. 10x protocol CG000520, Revision B (Deparaffinization, H&E staining and Imaging prior to Library Construction and CytAssist)
  

2. Additional Protocols/Resources
https://www.10xgenomics.com/support/spatial-gene-expression-ffpe
TITLE: Viral purification from bacterial culture
AUTHORS: sarah.schulz

[DESCRIPTION]
Protocol for the purification of viral particles from bacterial liquid culture

[STEPS]
1. Centrifuge 40 ml of bacterial culture infected with phage at 6000 g for 30 min, remove the supernatant and filter it through a 0.45 μm syringe filter
2. Centrifuge the filtrate at 35 000 g for 4 h, remove the supernatant and re-suspend the pellet in 600 μl SM buffer
3. Add 2 μl of DNAse I and 20 μl of 10x DNAse buffer and incubate at 37°C for 1.5 h
4. Incubate sample at 65°C for 30 min to inactivate DNAse I
5. Add 10 μl of 20 % SDS and 40 μl of Proteinase K (20 mg/ ml) and incubate at 37°C for 1 h
6. After the incubation, mix the sample with an equal amount of phenol:chloroform:isoamyl pH 8.0 (25:24:1) alcohol in a phase lock gel light tubes and centrifuge at 12 000 g for 5 min
7. Add 600 μl more of the phenol:chloroform:isoamyl alcohol to the tube and centrifuge at 12 000 g for 5 min
8. Transfer the aqueous phase to a new tube and add 1200 μl cold 100% ethanol
9. Incubate sample overnight at -80°C, and then centrifuge at 16 000 g for at 4°C for 1 h
10. Remove the supernatant and re-suspend the pellet in 100 μl TE buffer
11. Store at 4°C
TITLE: Complete Medium or Complete Medium Xylose  (from Leach, Lang and Yoder 1982)
AUTHORS: Megan Mcdonald

[DESCRIPTION]
For the growth and maintenance of Cochliobolus carbonum and Cochliobolus victoriae

[STEPS]
SECTION: Make Micronutrients Solution
1. 9 mg  H3BO3
58.5 mg CuSO4 *5H2O
1.95 mg KI (Potassium Iodine)
9 mg MnSO4
7.6 mg NaMoO4
822 mg ZnSO4 * 6 H2O
139.8 mg FeCl3 * 6H2O

in 300 mL ddH2O and filter sterilise

Citation:
Heterokaryosis and Parasexuality in the Fungus Ascochyta Imperfecta Author(s): K. E. Sanderson and A. M. Srb Source: American Journal of Botany , Jan., 1965, Vol. 52, No. 1 (Jan., 1965), pp. 72-81 Published by: Wiley Stable URL: https://www.jstor.org/stable/2439977

SECTION: Make 100x Salt Solutions A and B
2. 100X Salt Solution A

10g Ca(NaO3)2 * 4 H2O
100 mL ddH2O

Autoclave

Citation:
Leach, J., Lang, B. R. & Yoder, O. C. Microbiology128, 1719-1729, doi:https://doi.org/10.1099/00221287-128-8-1719 (1982).
SECTION: Make 100x Salt Solutions A and B
3. 100X Salt Solution B

2 g  KH2PO4 * 7 H2O
1.5g NaCl

100 mL H2O
pH 5.3

Autoclave

Citation:
Leach, J., Lang, B. R. & Yoder, O. C. Microbiology128, 1719-1729, doi:https://doi.org/10.1099/00221287-128-8-1719 (1982).
SECTION: Make Complete Medium (CM) or Complete Medium Xylose (CMX)
4. Complete Medium Base

10 g  glucose OR xylose (substitute glucose for xylose for CMX medium)
1 g Yeast Extract
0.5 g Acid-hydrolysed Casein
0.5 g Enzyme-hydrolysed casein
20g Agar
10 mL Salt A
10 mL Salt B

Make up to 1000 mL with ddH2O and Autoclave

After autoclaving add:
1 mL  sterilised micronutrient solution


Citation:
Leach, J., Lang, B. R. & Yoder, O. C. Microbiology128, 1719-1729, doi:https://doi.org/10.1099/00221287-128-8-1719 (1982).
SECTION: Complete Medium for Sporulation
5. Complete Medium Base

0.5 g  glucose OR xylose (substitute glucose for xylose for CMX medium)
20 g Sorbose
1 g Yeast Extract
0.5 g acid hydrolysed casein
0.5 g enzyme hydrolysed casein
20g Agar

10 mL Salt A
10 mL Salt B

Make up to 1000 mL with ddH2O and Autoclave

After autoclaving add:
1 mL  sterilised micronutrient solution
TITLE: RNA Extraction Protocol for Shorea
AUTHORS: mpfsum  

[DESCRIPTION]
RNA extraction protocol using CTAB method optimized for leaf and bud samples from Shorea curtisii.
Adapted from extraction protocol for Shorea beccariana (see attached publication).

[STEPS]
SECTION: Lysis
1. Weigh BUD 40 mg or LEAF 60 mg and transfer into a 1.5 mL tube.


SECTION: Lysis
2. Ground in CTAB buffer (3% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl pH 8.0, 0.2% b-mercaptoethanol) using tissuelyzer.
SECTION: Lysis
3. Incubate at 60 °C for 45 min. 

SECTION: Precipitation
4. Add 1 volume of chloroform.
Mix by vortex but not too vigorous.
Ensure that the two phases of the mixture are homogenized well.
SECTION: Precipitation
5. Centrifuge at5000 rpm, 1 min .
Transfer the supernatant into a new 1.5 mL tube.

SECTION: Precipitation
6. Add 2/3 volume of isopropanol.
Mix gently (turning the tubes upside down 2 min ).

SECTION: Precipitation
7. Centrifuge 5000 rpm, 5 min 
Discard solution carefully.
 

SECTION: Washing
8. Add 700 µL ethanol.
Mix gently (flicking the tube) until the pellet no longer stick to the tube.

SECTION: Washing
9. Centrifuge 5000 rpm, 5 min.
Discard solution carefully. 

SECTION: Washing
10. Air dry the pellet 1 min.
Repeat Washing if necessary.

SECTION: Resuspension
11. Resuspend the pellet in 50 µL RNAse-free water.
Flick the tube until the pellet is no longer visible.

If the pellet is difficult to dissolve, leave it for ~30 min before flicking again.
If it still does not dissolve, add more RNAse-free water AND adjust the subsequent steps accordingly.

SECTION: Genomic DNA Digestion
12. Removed gDNA using  .
Follow the manufacturer's protocol.

SECTION: Purification
13. Purification is done using  with minor adjustment to the protocol.
Add 350 µL RLT buffer.
Mix gently (flicking). 

SECTION: Purification
14. Add 250 µL absolute ethanol.
Mix gently (flicking).


SECTION: Purification
15. Transfer the solution into RNeasy Mini Spin column.
Centrifuge 10000 rpm, 15 s.
Discard the flow through.


SECTION: Purification
16. Add 700 µL RW1 buffer to the column.
Centrifuge 10000 rpm, 15 s.
Discard the flow through.

SECTION: Purification
17. Add 500 µL RPE buffer to the column.
Centrifuge 10000 rpm, 15 s.
Discard the flow through.

SECTION: Purification
18. Add 500 µL RPE buffer to the column.
Centrifuge 10000 rpm, 2 min.
Discard the flow through.
SECTION: Purification
19. Replace the collection tube.
Centrifuge 14000 rpm, 1 min to further dry the membrane.

SECTION: Purification
20. Replace the collection tube with a new 1.5 mL tube.
Add 50 µL RNAse-free water directly onto the membrane.
Incubate at Room temperature for 5 min.

SECTION: Purification
21. Centrifuge 10000 rpm, 1 min.
Store the extracted RNA in -20 °C or -80 °C for longer storage.

SECTION: Quality Assessment
22. Evaluate the RNA quality using Agilent 2100 Bioanalyzer.
TITLE: Treatment schemes and persistence in Colombian patients diagnosed with inflammatory arthritis after the failure of conventional disease-modifying antirheumatic drugs
AUTHORS: Jorge Machado Alba

[DESCRIPTION]
Introduction:
Inflammatory arthritis is related to disability, chronic pain, and premature
death. A lack of treatment persistence leads to poor control of symptoms, inflammation,
and joint damage, thereby worsening quality of life.
 
Objective: To
determine the effectiveness of antirheumatic drug treatment and the persistence
of use in Colombian patients diagnosed with rheumatoid arthritis (RA),
psoriatic arthritis (PsA), ankylosing spondyloarthritis (AS), and juvenile
rheumatoid arthritis (JIA) after the failure of conventional disease-modifying
antirheumatic drugs. (cDMARDs).
 
Methods: In this retrospective
descriptive study, patients who were diagnosed with RA, PsA, AS, or JIA; were
treated at a center specializing in rheumatological diseases; who started their
first treatment with biological DMARDs (bDMARDs) or tofacitinib between
February 2016 and December 2019; and who were followed up until the
discontinuation of treatment or 24 months were evaluated.
 
Results: A total of 426
patients were included; 78.8% were women, and the mean age was 50.2±14.1 years.
The majority had a diagnosis of RA (71.8%). A total of 89.9% had received
cDMARDs, and 77.2% had received glucocorticoids. The most frequently initiated
bDMARDs were rituximab (31.2%), etanercept (23.0%), and adalimumab (14.6%). A
total of 80.3% of patients received concomitant cDMARDs. During an average
follow-up of 635.2±189.6 days, 12.9% of patients had changes in their treatment
regimen, 26.3% had interruptions in their treatment regimen, and 23.9%
discontinued bDMARDs or tofacitinib. Patients who received concomitant cDMARDs
were more likely to continue their biological therapy (odds ratio: 8.50; 95% confidence
interval: 3.49-20.73; p<0.001).
 
Conclusions: Follow-up
evaluation of this group of patients revealed that they were treated mainly
with non-TNF-α inhibitors associated with cDMARDs, and a low proportion of the
patients had changes in therapy, although one-quarter had treatment interruptions
or discontinuations.

[STEPS]
TITLE: Intracardiac perfusion with fixative for ultrastructural neuroanatomical studies
AUTHORS: Janet R Keast, Peregrine Osborne

[DESCRIPTION]
This protocol is suitable for preserving tissues for ultrastructural neuroanatomical studies of peripheral nerves, ganglia, spinal cord or brain in adult rats. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation.

[STEPS]
SECTION: Perfusion
2. Induce anesthesia by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).
SECTION: Perfusion
3. After opening the chest cavity, inject the left ventricle with mixture of 0.25 ml heparin (5000 IU/ml) and 0.5 ml 1% sodium nitrite.
SECTION: Perfusion
4.  A needle connected to tubing (T-connector to prewash and perfusion solutions), and a peristaltic pump (setting: 50 ml/min) is then inserted into the left ventricle and clamped into place using hemostats. Make a small incision in the right atrium to drain blood and perfusate during the procedure.
SECTION: Perfusion
5.  Perfuse with pre-wash solution until the fluid flowing from the right atrium is clear, the liver and extremities are pale (typically 2-3 min). 
SECTION: Perfusion
6.  Perfuse with fixative for 15-20 minutes, by which time the organs have stiffened and the neural tissues will be well preserved. 
SECTION: Perfusion
7. Dissect tissues required for analysis and place in fixative for a further 18-24h at 4C.
SECTION: Post-perfusion
8. Wash tissues with phosphate-buffered saline (PBS; 0.1 M, pH7.2), 3 x 30 min washes.
SECTION: Post-perfusion
9. Store in PBS at 4C until processed further for embedding and electron microscopy.
SECTION: Preparation for perfusion
1. Make up the following solutions:

Perfusion prewash solution: To 300 ml 0.9% sodium chloride (w/v) add 3.75 ml 1% sodium nitrite (w/v) and 0.11 ml heparin (5000 IU/ml). This is made up immediately prior to use.

Perfusion fixative: 2% paraformaldehyde and 1.25% glutaraldehyde in 0.1M phosphate buffer, pH 7.4. The buffered paraformaldehyde solution is made up no longer than 48h prior to use, stored at 4C and brought to room temperature on the day of perfusion. Glutaraldehyde is added to this solution on the day of the perfusion.