eamag/protocols-with-steps huggingface dataset:
TITLE: Golden Gate Assembly
AUTHORS: Amanda T Alker, Nicholas J Shikuma

[DESCRIPTION]
Protocol for golden gate assembly of modular plasmids from Yeast Toolkit, Bee Toolkit and Marine Modification Kit platforms.

[STEPS]
SECTION: Day 1
1. Streak out the plasmid parts that will be used in the assembly onto LB agar plates with the appropriate antibiotics (Chloramphenicol 100µg/mL for Type 1-7 parts; Kanamycin or Gentamicin 100µg/mL for Type 8 backbone parts). Incubate overnight at 37ºC. 
SECTION: Day 2
2. Inoculate a single colony into 25mL of LB plus antibiotics in the late afternoon/early evening. Incubate overnight at 37ºC while shaking at 200rpm.
SECTION: Day 3
3. Spin the culture at 5000g for 20 minutes. Remove the supernatant, resuspend the pellet in 1mL water. Perform a plasmid miniprep (Zyppy miniprep or Omega E.Z.N.A. Plasmid Mini Kit II) following the standard kit protocols. 
SECTION: Day 3
4. Measure the Plasmid DNA concentration on a spectrophotometer. 
SECTION: Day 3
5. Perform the Golden Gate Assembly:
Dilute backbone plasmid parts and add 10fmol to the reaction
Dilute the insert plasmid parts and add to 20fmol of each insert to the reaction
Add 2µL T4 Ligase Buffer (Promega)
Add 1µL T4 Ligase
Add 1µL BsaI-HF_V2 or BsmBI-HF endonuclease
Add X water up to a 20µL reaction

 


SECTION: Day 3
6. Run the thermocycler program for BsaI/BsmBI as follows:
 

 

 

SECTION: Day 4
7. Dilute 2x or electroporate 2µL of the GGA directly into electrocompetent cells (i.e. SM10pir, S17pir). Recover 1+ hours and plate on LB Agar media containing the correct antibiotic concentrations. Incubate at 37ºC overnight. 
SECTION: Day 5
8. Screen colonies for correct insert and perform colony PCR with primers spanning assembly junctions. 
SECTION: Day 5
9. Streak out clones that yield a band onto LB Agar media containing the correct antibiotic concentrations and incubate at 37ºC overnight. 
SECTION: Day 6
10. Inoculate a single colony into 25mL LB broth containing the correct antibiotic concentrations and incubate at 37ºC overnight. 
SECTION: Day 7
11. Store overnight culture in cryovials for long term storage. Add 500µL of culture to 500µL of 50% glycerol and store in -80ºC freezer. 
SECTION: Day 7
12. With remaining culture, Centrifuge at 5000g for 20 minutes. Remove the supernatant, resuspend the pellet in 1mL water. Perform a plasmid miniprep (Zyppy miniprep or Omega E.Z.N.A. Plasmid Mini Kit II) following the standard kit protocol. Elute with water. 
SECTION: Day 7
13. Measure the Plasmid DNA concentration on a spectrophotometer. 
SECTION: Day 7
14. Send the miniprepped plasmid for Sanger or Oxford Nanopore long-read plasmid sequencing to confirm the construct. Store the remaining miniprep for downstream applications (i.e. shuttle into different electrocompetent cell for conjugation).
TITLE: Plate reader setting
AUTHORS: Junyan Qian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A brief guideline of setting the plate reader to measure the sfGFP production.</div></div>

[STEPS]
?. Create a new protocol in PerkinElmer by clicking start wizard in Victor Workstation. Name it as the substance you want to measure. In our case sfGFP, select a folder to save the protocol. Click Next>.
?. Select the plate you are using for the measurement, it allows the plate reader to have a default setting of the position of each well according to the plate you chose, we used NUNC 384. Click Next>.
?. Select or create the label we are using for the measurement, the label is usually named as the substance you are testing it gives setting for lamp, filter and counting time for the measurement.Click Label (the fourth icon)on the right navigation bar to add labels and search in Fluorometry tab to see if there is already existing sfGFP label.  If yes, choose that label, skip the sub-steps, and click Next> to proceed to step 4.
?. Assuming there is no existing sfGFP label,  in Fluorometry tab Add a new label, name it sfGFP, click OK.
?. Set CW-Lamp Energy: 17000 (depends on excitation wave length)Set CW-Lamp Control: Stabilized EnergySet CW-Lamp Filter: F485 (Excitation filter)Set Emission Filter: F535Set Emission Aperture: Normal (4mm in diameter)Set Counter position: BottomSet Counting time: 0.2 sClick OK
?. Select the label you just create(sfGFP) click OK then Next>
?. Select the wells you are going to measure, (each time when you start a measurement, check and modify the wells you are measuring). Click Next>. If there is no additional information or description you want to add, click Finish to start the measurement.
?. A shaking procedure is recommended to be added before measurement.This step tells how to include shaking.
?. In PerkinElmer 2030 Manager interface:Select Protocol editor(third icon on the second tab row), Click Measurement tab,     On the right there is a column of events, Click Shake.
?. Set Shaking duration: 1.0sSet Shaking speed: NormalSet Shaking diameter: 0.10mmSet Shaking type: LinearSet Repeated operation : NoClick OK
?. Because we want to shake it before each measurement,select Shake and click the up-pointing arrow on the left make sure it's on the top of sfGFP label.
?. Our PURE expression is carried on in  , set the temperature to
37 °C
37 °C
?. Wait until the plate reader reaches the desired temperature, click Start icon to start the measurement. Once a protocol is created it can be used for the same measurement next time, but don't forget to modify the wells you are measuring.
?. In PerkinElmer 2030 Manager interface: Select Temperature tab, Turn on Plate heating option, Set target temperature to  , Click Apply
37 °C
TITLE: Mini culture slants for long term storage of fungi
AUTHORS: You Li, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to store fungi in slants.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: SOP – 3-step protein fractionation from fly heads
AUTHORS: ALFONSO.M.PENA

[DESCRIPTION]
SOP – 3-step protein fractionation from fly heads

[STEPS]
SECTION: Protocol:
7. Collect 20-40 fly heads via Snap Freeze method into a Biomasher tube and transfer tubes to CTRND on dry ice.
SECTION: Protocol:
8. Turn on Sorvall ultracentrifuge and set temperature to 4degC, place rotor inside and turn on vacuum to allow internal temperature to set.
SECTION: Protocol:
9. Add 50uL (2.5uL/fly head) of TBS with PPI to heads in Biomasher II tube and homogenize for 1.5 min with a hand-held automatic homogenizer and Biomasher pestles.
SECTION: Protocol:
10. Briefly spin down in tabletop centrifuge to get the liquid to the bottom of the tube.
SECTION: Protocol:
11. Sonicate at 35% with 5 one-second pulses for tissue to break down/dissolve. Skip this step for ‘non-sonicated’ extractions.
SECTION: Protocol:
12. Clarify by centrifugation in a tabletop centrifuge (2,100g) for 15 seconds.
SECTION: Protocol:
13. Move entire liquid sample into ThermoScientific 5ml microtube WX und MX (Cat #314352H01) before centrifuging.
SECTION: Protocol:
14. Centrifuge the supernatant at 100,000 g (29,900 rpm) for 30 min at 4°C using the chilled rotor.
SECTION: Protocol:
15. Remove and save Supernatant into new labeled collection Eppendorf (TBS-soluble fraction).
SECTION: Protocol:
16.  Wash the pellet by adding 50uL of TBS with PPI and centrifuging at 100,000 g for 15 min at 4°C. Discard the supernatant.
SECTION: Protocol:
17. Homogenize pellet with 30uL (1.5ul/fly head) of solvent with PPI with hand-held homogenizer.
SECTION: Protocol:
18. Centrifuge at 100,000 g for 30 min at 4°C and save supernatant into new labeled collection Eppendorf (_-soluble fraction).
SECTION: Protocol:
19. Wash the pellet by adding solvent 2 with PPI and centrifuging at 100,000 g for 15 min at 4°C. Discard the supernatant.
SECTION: Protocol:
20. Resuspend pellet in 30ul (1.5ul/fly head) in 8M urea/5% SDS buffer with PPI by sonicating with 3 one-second pulses until it is fully resuspended. Label and save (insoluble fraction)
SECTION: Protocol:
21. Perform BCA. If strapped on time, samples can wait to be quantified and stored at -80C.
SECTION: Reagents Needed:
1. Protease and Phosphatase Inhibitors (cOmplete 04 693 124 001, PhosphoSTOP 04 906 837 001)
SECTION: Reagents Needed:
2. Urea – Fisher Bioreagents #BP169-500
SECTION: Reagents Needed:
3. SDS – Fisher Bioreagents #BP166-500
SECTION: Reagents Needed:
4. RIPA Buffer (PierceTM, Thermo Scientific 89900)
SECTION: Reagents Needed:
5. NP-40 (Nonidet® P 40 Substitute, Sigma, Cat #74385)
SECTION: Reagents Needed:
6.
TITLE: Sample submission for LC-MS BioMS core facility at the University of Manchester
AUTHORS: ronan.ocualain

[DESCRIPTION]
Submitting samples to the BioMS core facility.

[BEFORE_START]
Peptides should be dried down, use the speed vac in lab B2075 for this purpose.
Instructions are here

[GUIDELINES]
Peptides should be dried down, use the speed vac in lab B2075 for this purpose.
Instructions are here

[STEPS]
1. Place the dried down peptides into the drawer in B2071, either in a labelled bag (<20), or box if you are submitting a large batch of samples.

On the label, you must include 
PPMS order number (not project), 
number of samples, 
and any other important details, such as run length, 
sample manifest (i.e. which order you wish the data to be acquired in, if minimization of carryover is important),
and whether the samples have been prepared in S-trap lysis buffer, or another buffer (e.g. urea, triton etc). 

Don’t forget to include your lab group/PI, as this is the folder within which your data files will be stored on the server.
TITLE: DNA extraction NucleoSpin Tissue INRAE eWHALE 
AUTHORS: Teddy Urvois, Anne-Laure Besnard, Erwan Quéméré

[DESCRIPTION]
This DNA extraction protocol aims at extracting total genomic DNA from filtered seawater samples suspended in Tris-EDTA-SDS Buffer.

Our protocol mostly follows the NucleoSpin Tissue protocol (qr.mn-net.com/qr/(IFU)740952), section 5 (Standard protocol for human or animal tissue and cultured cells). It starts at step 3, with 200µL of sample, and adds 25µL Proteinase K to the original step. To maximize DNA concentration, Buffer BE is heated at 70°C, and elution is repeated twice with the same 100µL of Buffer BE with 3 min incubation.

[BEFORE_START]
Sample collection and preparation:
Seawater was filtrated using Sylphium pods with 0.45µm filters. Using a syringe, water was removed and filters dried.
To lyse cells and preserve DNA, 3mL of Tris-EDTA-SDS Buffer was added to each Sylphium pod. Sylphium pods were freezed quickly after.

Lab work:
Prepare Buffer B5 and Proteinase K.
Put all equipment (micropipettes, disposable tips, microtubes, columns, ethanol, B3, BW and B5) in UV cabinet and
light UV for 20 mins.
Set incubator to 70°C and heat Buffer BE to 70 °C.
Vortex and spin samples.

[STEPS]
SECTION: Lyse sample
1. Add 200 µL of   to a microcentrifuge tube.
Add 200 µL Buffer B3 and 25 µL Proteinase K solution.
Vortex vigorously.
Incubate at 70 °C for 10 min.
Vortex briefly.
SECTION: Adjust DNA binding conditions
2. Add 210 µL ethanol (96 – 100 %) to the sample and vortex vigorously.
SECTION: Bind DNA
3. For each  , place one NucleoSpin Tissue Column into a Collection Tube.
Apply the   to the column.
Centrifuge for 1 min at 11,000 x g.
Discard Collection Tube with flowthrough and place the column in a new Collection Tube.
SECTION: Wash silica membrane
4. Add 500 µL Buffer BW.
Centrifuge for 1 min at 11,000 x g.
Discard flowthrough and place the column back into the Collection Tube.
Add 600 µL Buffer B5 to the column and centrifuge for 1 min at 11,000 x g.
Discard flowthrough and place the column back into the Collection Tube.
SECTION: Elute highly pure DNA in two steps with Buffer BE heated at 70°C
5. Place the NucleoSpin Tissue Column into a 1.5 mL microcentrifuge tube and add 100 µL Buffer BE.
Incubate at room temperature for 3 min.
Centrifuge 1 min at 11,000 x g.
Add the same 100 µL Buffer BE back in the NucleoSpin Tissue Column.
Incubate at room temperature for 3 min.
Centrifuge 1 min at 11,000 x g.
Discard NucleoSpin Tissue Column.
TITLE: Fractionation of synaptosomes
AUTHORS: Chuyu Chen, Ciarra Smith, Loukia Parisiadou

[DESCRIPTION]
This protocol details a step by step method to prepare pure fractions of synaptosomes for biochemical analysis. 

[STEPS]
1. Pre-chill the homogeniser and buffers on ice. Weigh the tissue
2. Add 4x volume of ice-cold homogenizing buffer to glass homogenizers
3. Apply 12 strokes of even tension with the homogeniser rod
4. Transfer to 1.5ml tube (original tube)
5. Determine which tissue has the smallest volume and use that volume for all samples
6. Centrifuge at 1,000g for 10mins
7. Transfer the supernatant into fresh 1.5ml tube and spin at 12,500g for 15mins
8. Discard supernatant carefully
9. Resuspend pellet in 1ml of H-buffer, transfer to fresh glass homogenizer and mash for 6 strokes even tension
10. In polypropylene/ultra-clear tube add 5ml of 1.2M sucrose
11. Slowly and steadily add in 5ml of 0.8M sucrose to create clear/distinct gradient
12. Overlay sample on top of gradient
13. Weigh sucrose gradient tubes (add H-buffer if weight is off by more than 0.05g)
14. Centrifuge with slow accleration and zero brake in 4 degrees at 23,600g for 70 mins in a SW41 rotor
15. Using syringe, extract synaptosome layer between 0.8M and 1.2M sucrose and transfer into fresh 1.5ml tubes
16. Plate sample with appropriate dilutions into 24-well plate (dilute with H-buffer or PBS)
17. Spin in 4°C, max speed ~4,000 rpm for 20mins
18. Remove liquid and fix in 4% PFA for 20mins on rocker/shaker for 20mins
19. Wash x2~3 with PNBS
20. Either cover and place in fridge or move directly to immunofluorescence protocol
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Este protocolo describe los pasos para propagar <em>in vitro</em> hongos tipo <strong>terraza</strong> (Ver <a href="https://www.protocols.io/view/cultivo-in-vitro-de-hongos-tipo-sombrero-ng4dbyw" target="_blank">este</a> protocolo para hongos tipo <strong>sombrero</strong>). Este protocolo ha sido desarrollado bajo la guia de Phil Ross (Mycoworks; https://www.mycoworks.com).</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tyheme6.jpg" data-src="https://s3.amazonaws.com/pr-journal/tygeme6.jpg" data-ofn="va4-2.jpg" /></p>

[BEFORE_START]
<p>Al muestrear se debe recolectar todo el cuerpo fructífero que sea posible, en particular se debe intentar sacar parte del micelio que se encuentra dentro de la madera.  </p>
<p> </p>
<p>Las muestras deben ser cultivadas lo antes posible luego de su recolección para aumentar las probabilidades de éxito. Si bien la duración del hongo antes de su descomposición dependerá de las condiciones de almacenaje y el hongo en sí, se recomienda realizar los cultivos dentro de las 24 horas siguientes a la recolección.  </p>
<p> </p>
<p>El cultivo de las muestras debe hacerse en condiciones de esterilidad.</p>
<p> </p>
<p>Todas las placas deben ser correctamente selladas con parafilm para evitar su deshidratación.   </p>
<p> </p>
<p>Finalmente, cabe recordar que si bien, muchos de los hongos que se puedan recolectar son cultivables por este método, existen varios que serán difícilmente cultivable o no podrán ser cultivados. Dado lo anterior, es importante sacar siempre varias muestras y seguir los pasos con la mayor precisión posible para aumentar las probabilidades de éxito.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Food safety knowledge, attitudes, and eating behavior  under the global coronavirus pandemic
AUTHORS: Zhe  Liu

[DESCRIPTION]
The objective of this lab protocol was to evaluate the relationships among food safety knowledge, attitude and eating behavior of consumers during lockdowns in the advent of the COVID-19 pandemic. A total of 157 respondents completed the online survey using a structured questionnaire worldwide. Overall, the respondents exhibited good attitude and good knowledge towards public health including food safety especially on the importance of social distancing, mask wearing, well-balanced diet, physical exercise and personal hygiene, such as hand washing during the pandemic lockdowns. A Structural Equation Modeling (SEM) was established to test the relationships among food safety knowledge, attitude and behavior under the pandemic conditions. Results showed that attitude towards food safety under the coronavirus pandemic and lockdowns positively affected the eating behavior of the respondents, which exhibited a high β (0.686) among the variables tested (p<0.05). Food safety knowledge was apparently not affected by the food safety behavior of the respondents.

[STEPS]
TITLE: ATP_microplate_settings
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ATP assay- Using microplate reader</div></div>

[STEPS]
?. [ATP assay- Using microplate reader]
Sign up for the microplate when planning the experiment and power on before ~15 minutes of use
?. [ATP assay- Using microplate reader]
Mix your samples and reagents in the microplate following the manufacturer’s protocol
?. [ATP assay- Using microplate reader]
Start a new protocolSelect Plate typeCheck box- Use LidSet temperatureShake        1. Shake mode –eg. Orbital        2. Shake duration-eg. 15 secondsRead        1. Shake mode –eg. Orbital        2. Shake duration-eg. 15 seconds        1. Detection method- Luminescence        2. Read type- Endpoint/Kinetic        3. Optics type- Filters --------Filter set- Green; Integration time- 1sec, click full plate and select wells you want to read
TITLE: Transformation of Plasmid into Competent E. coli Cells (e.g., DH5α)
AUTHORS: Hussain Zubair

[DESCRIPTION]
This is a detailed experimental protocol for the transformation of plasmid DNA into competent E. coli cells, specifically using the DH5α strain. The protocol encompasses the entire process, from the preparation of LB agar plates supplemented with ampicillin to the final inoculation of transformed cells in LB broth. Key steps include the careful thawing of competent cells, the addition of plasmid DNA, a precise heat shock treatment at 42°C for enhanced plasmid uptake, and subsequent recovery and plating of the cells. The methodology is designed to maximize the efficiency of transformation, crucial for subsequent molecular biology experiments such as gene cloning and expression analysis. This protocol serves as a fundamental guide for researchers in the field of molecular genetics and microbiology.

[STEPS]
SECTION: Preparation of LB Agar Plates with Antibiotics
1. Prepare LB (Luria-Bertani) agar plates.
Once the agar has cooled but not solidified, add ampicillin at a ratio of 1:1000 to the agar.
Pour the LB agar into 90 mm Petri dishes and allow to solidify. 

SECTION: Thawing of Competent Cells
2. Retrieve the competent E. coli cells (e.g., DH5α strain) from the -80 °C freezer.
Thaw the cells slowly on ice to ensure they remain in liquid form. 10 min 


SECTION: Addition of Plasmid
3. Add 1 µL  of the plasmid solution to the competent cells. The volume may vary depending on the plasmid's concentration.
Incubate the mixture on ice for 30 min to allow the cells to fully absorb the plasmid. 


SECTION: Heat Shock Treatment
4. Subject the cell-plasmid mixture to heat shock in a water bath set at 42 °C  for exactly 70 s.
This step facilitates the uptake of the plasmid by the cells.




SECTION: Cooling Period (Stabilizing the Cells)
5. Immediately after the heat shock, place the mixture on ice for 2 min to stabilize the cells. 

SECTION: Plating on LB Agar
6. Gently spread the competent cell-plasmid mixture onto the prepared LB agar plates with ampicillin.
Incubate the plates at 37 °C  2 min to allow for bacterial growth and colony formation. 

SECTION: Further Inoculation
7. The following day, select a single colony from the agar plate.
Inoculate the colony in LB broth for further growth and experimentation as required
TITLE: Protocol for selective growth of Acetobacter pomorum (Ap) and Lactobacillus plantarum (Lp)
AUTHORS: Lúcia Serra, Sílvia Henriques, Carlos Ribeiro

[STEPS]
?. - Inoculate fresh mannitol (200 ml) and MRS (10 ml) media (less than a week old) with a single colony of Ap and Lp, respectively. Incubate Ap at 30 ºC, 180 rpm; and Lp at 37 ºC, without shaking.
?. - Dilute 250 µl Lp culture in fresh 10 ml MRS media. - Ask for MRS, MRS + Kanamycin (50 µg/ml) and MRS + Ampicillin (10 µg/ml) plates. The plates should be as fresh as possible - don’t use plates more than one week old.
?. - Measure OD (600 nm) of each culture.- Dilute the highest OD (should be Lp) to the same value of the lowest OD in liquid media.For example, if Lp has       an OD of 4.6 and Ap, 0.61, dilute Lp in 1 ml MRS by mixing 132 µl Lp culture and 868 µl MRS to obtain cultures with equivalent numbers of Lp and Ap cells. - Prepare adequate Ap and Lp dilutions in PBS 1X and inoculate 50 µl of each culture (Ap and Lp) in each plate:Two MRS plates One MRS + Kan plate One MRS + Amp plate- Use sterile glass beads to spread the cells and incubate the plates (for 48 hours) as follows: One MRS plate at 37ºC (optimal growth of Lp) One MRS plate at 30ºC (optimal growth of Ap) MRS +  Kan plate at 37ºC MRS +  Amp plate at 30ºC - Prepare adequate Ap and Lp dilutions in PBS 1X and inoculate 50 µl of each culture (Ap and Lp) in each plate:Two MRS plates One MRS + Kan plate One MRS + Amp plate- Use sterile glass beads to spread the cells and incubate the plates (for 48 hours) as follows: One MRS plate at 37ºC (optimal growth of Lp) One MRS plate at 30ºC (optimal growth of Ap) MRS +  Kan plate at 37ºC MRS +  Amp plate at 30ºC - Use sterile glass beads to spread the cells and incubate the plates (for 48 hours) as follows: One MRS plate at 37ºC (optimal growth of Lp) One MRS plate at 30ºC (optimal growth of Ap) MRS +  Kan plate at 37ºC MRS +  Amp plate at 30ºC
?. - Verify if Ap and Lp have grown in the following conditions: Ap should only grow in MRS (either 37ºC or 30ºC) and MRS + Amp Lp should only grow in MRS (either 37ºC or 30ºC) and MRS + Kan
?. For separate plating of Ap and Lp, the same steps are performed, but 50 µl of each culture are plated in each plate of MRS, MRS + Kan and MRS + Amp, separately. The total number of plates will double.
TITLE: De-novo assembly of Xanthomonas genomes from Illumina NovaSeq reads
AUTHORS: David J Studholme, Jamie Harrison

[DESCRIPTION]
This protocol describes the de-novo assembly of Xanthomonas genome sequences from short-read genomic shotgun sequencing data. It includes quality control of the raw sequence reads, assembly and finally polishing of the assembly based on alignment of reads against the preliminary assembly.

[STEPS]
5. Bibliography


 

 

 

 

 

2. Perform quality-based filtering and adapter trimming using fastp.

 

 

3. Perform de-novo assembly using SPAdes.


 

4. Polishing with Pilon

 

 

 

 

 

 

 

 



 



1. Software pre-requisites.
TITLE: Nissl (Cresyl Violet) staining
AUTHORS: Christiana.bjorkli  , Karoline Hovde, Bruno Monterotti

[DESCRIPTION]
Nissl (Cresyl Violet) staining protocol 

[STEPS]
1. Dehydrate sections – 10 dips in each: 50, 70, 80, 90, 100, 100, and 100% ethanol 
2. Leave in xylene for 2min 
3. Rehydrate sections – 10 dips in each: 100, 100, 100, 90, 80, 70, and 50% ethanol 
4. Quick wash in running water
5. Leave sections in Cresyl Violet for 3min on shake
6. Leave sections in Citric Acid for 1min 
6.1. Dip in running water 3x 
6.2. Dip in Citric Acid 3x 
6.3. Dip in running water 3x 
7. Leave sections in Cresyl Violet for 2min on shaker 
8. Repeat step 6 
9. Dehydrate sections – 10 dips in each: 50, 70, 80, 90, 100, 100, and 100% ethanol
10. Leave in Xylene for at least 5min 
11. Coverslip with Xylene + Entellan