eamag/protocols-with-steps huggingface dataset:
TITLE: Immunofluorescence staining of myenteric and submucosal plexuses
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details myenteric and submucosal plexus immunostaining.

[STEPS]
SECTION: Procedure
1. 
 
Cut out two small regions of each plexus sheet in PBS at 4 °C.

SECTION: Procedure
2. Add each tissue section to separate wells in a 96 well dish.
SECTION: Procedure
3. Add 10% normal donkey serum (Jackson Immunoresearch, Cat #017-000-121; West Grove, PA) and 1% Triton-X in PBS. Leave in blocking solution for 60 min at Room temperature on a rotator.
SECTION: Procedure
4. Incubate the tissue 60 min at Room temperature on a rotator with primary antibodies in 10% normal donkey serum, 1% Triton-X in PBS.
SECTION: Procedure
4.1. Stain sections with antibodies against tyrosine hydroxylase (TH) (1:500, Millipore-Sigma, Cat # AB152; Burlington, MA) and CD3 (1:400, Bio-Rad Laboratories, Cat #MCA2690; Hercules, CA), ANNA1 (1:32,000; kind gift by the Gershon laboratory (Margolis et al., 2016)), Iba1 (1:500, WAKO, Cat # 019-19741; Richmond, VA), CD68 (1:1000, Abcam, Cat # ab53444; Waltham, MA).
SECTION: Procedure
5. On Day 2, remove the primary antibody solution from each well and wash.
SECTION: Procedure
6. Incubate in secondary antibody (1:1000) in blocking solution for 120 min at Room temperature, covered on a rotator.

SECTION: Procedure
7. Wash again.
SECTION: Procedure
8. Mount on slide with vectashield medium with DAPI
SECTION: Procedure
9. Imaging:
SECTION: Procedure
9.1. For imaging enteric neurons, for each plexus collect 2-3, 2x2 tile z-stack 640.17x640.17 µm confocal images at 20x magnification.
SECTION: Procedure
10. Analysis:
SECTION: Procedure
9.2. For imaging macrophages, for each plexus collect 2-3, 2x2 tile z-stack 390.09 x 390.09 µm confocal images at 20x magnification.
SECTION: Procedure
10.1. Count the number of ANNA1+, TH+ cells, and IBA1+ cells for each stacked image using Fiji.
SECTION: Procedure
10.2. Within the SP, threshold the TH+ signal, then analyze mean fluorescent intensity (MFI) and the area of the TH signal. Keep the thresholding consistent across each image, animal, and condition within each experiment.
SECTION: Procedure
10.3. Within each animal, sum the the number of ANNA1+ , TH+ cells, and IBA1+ cells across all images separately then divide by the acquisition area.
SECTION: Procedure
10.4. For each experiment, normalize to the CFA only condition.
SECTION: Procedure
5.1. Wash with PBS-Tween (0.1%) for 10 min. (1/3)

SECTION: Procedure
5.2. Wash with PBS-Tween (0.1%) for 10 min. (2/3)
SECTION: Procedure
5.3. Wash with PBS-Tween (0.1%) for 10 min. (3/3)
SECTION: Procedure
7.1. Wash for 10 min each in 0.1% PBS-Tween. (1/3)

SECTION: Procedure
7.2. Wash for 10 min each in 0.1% PBS-Tween. (2/3)
SECTION: Procedure
7.3. Wash for 10 min each in 0.1% PBS-Tween. (3/3)
TITLE: Foreskin Tissue DNA Extraction
AUTHORS: Brandon Maust

[DESCRIPTION]
Extracts total DNA from foreskin tissue, as used in Maust et al. 2022

[STEPS]
SECTION: Preparation
1. Add steel bead to 1.5 ml tube
SECTION: Preparation
2. Add 800 µL CD1 to tube
SECTION: Preparation
3. Weigh tube (with bead and solution)
SECTION: Tissue
4. Dissect approximately 25 mg (3 mm3) piece of tissue
Fragment tissue with scalpel
Add tissue to tube (with bead and solution)
SECTION: Tissue
5. Weigh tube and calculate net tissue weight.
SECTION: Pre-robot processing
6. Process tubes on TissueLyzer II, 2 min at 30 Hz
Rotate block 180º and repeat
SECTION: Pre-robot processing
7. Centrifuge tubes briefly to collapse foam
SECTION: Pre-robot processing
8. Spin PowerBead Pro plate to ensure beads are settled at the bottom
SECTION: Pre-robot processing
9. Add contents of each specimen tube (except steel bead) to a PowerBead plate well
SECTION: Pre-robot processing
10. Add 5 µL of 20 mg/ml proteinase K to each well
SECTION: Pre-robot processing
11. Seal plate with film 
Vortex briefly to mix
SECTION: Pre-robot processing
12. Incubate plate at 65º C for 60 min or until tissue is mostly digested
SECTION: Pre-robot processing
13. Process plate on TissueLyser II, 5 min at 25 Hz
Rotate plate 180º and repeat
SECTION: Pre-robot processing
14. Centrifuge plate at 3000 x g for 7.5 min
SECTION: Pre-robot processing
15. Transfer approximately 350 µL supernatant from each well to fresh S-block using well-vator
SECTION: Pre-robot processing
16. add 300 µL of solution CD2 to each well and mix thoroughly by pipetting
SECTION: Pre-robot processing
17. Seal the plate with film
Centrifuge at 3000 x g for 7.5 min at room temperature
SECTION: Pre-robot processing
18. Avoiding the pellet, transfer 500 µL of supernatant to fresh S-block using well-vator
SECTION: QIAcube
19. Follow DNeasy 96 PowerSoil Pro Protocol for QIAcube HT (page 17 onward)
Elute in maximum volume (120 µL)
Incude vacuum performance check
SECTION: QIAcube
20. Seal plate and freeze at -20ºC
TITLE: Neural aggregate formation
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Harvest iPSCs for neural aggregate formation when iPSCs have reached 75- 85% confluency. Aspirate medium and rinse with  of DPBS.
2 ml
?. Add  of Accutase. Incubate at for . Gently tap plate to dislodge cells.
1 ml
?. Dilute Accutase with  of DMEM/F12 medium and collect cell suspension in 15ml conical tube.
4 ml
?. Centrifuge cells at 750 rpm for . Then carefully aspirate medium from iPSC pellet.
?. Add  of neural induction medium to iPSC pellet. Using a hemacytometer, count iPSCs. Adjust volume of iPSC suspension to 450-650,000 cells/mL using neural induction medium supplemented Rock inhibitor (10 μM final).
3 ml
?. Add of iPSC suspension per well to a v-bottom 96-well plate.
100 µl
?. Centrifuge plate at 750 rpm for  to sediment iPSC into spheres.
?. Incubate cells at , 5% CO2 and 95% humidified chamber for . After 24 hrs, carefully remove all medium from well and replace with  per well of Neural Induction Medium.
100 µl
Do not disturb or break apart spheres. The spheres are very delicate at this stage.
?. Incubate neurospheres in 96 well plate for . Perform half volume medium changes daily (removed  and replace with ).
50 µl
50 µl
TITLE: pCP20 transformation to remove Kanamycin cassette
AUTHORS: Saul   Moore

[DESCRIPTION]
Transformation with pCP20 to remove the Kanamycin cassette from Keio E. coli BW215113 single gene deletion mutants - Cassandra Backes (Host-Microbe Co-Metabolism Laboratory, MRC-LMS)

[STEPS]
1. Make buffer and broth the day before and keep them in the fridge
2. Day before: Grow O/N cultures of the strains of interest. Can also prepare buffers (see materials). 
3. Dilute overnight culture in LB broth at an OD = 0,2 and grow until OD595nm = 0.5 – 0.8 (mid-log phase). 
UNTIL NOW, KEEP CULTURES ON ICE
4. Spin down culture(s): transfer first to 15 mL falcon, spin 4500 RPM, 10 min, 4°C
5. In the meantime, label 2 mL tubes with strain names and add 80uL TSS buffer and 1 - 5 uL of pDNA (50-75ng). vortex and keep on ice for 10 min. 
6. Remove supernatant from 14 mL tubes and resuspend pellet in 1mL TSS broth(cold). Resuspend gently and keep on ice. 
7. Then transfer cells to a 2 mL tube. 
8. Add 200uL of bacterial culture in TSS broth to the 2 mL tubes containing pDNA in TSS buffer, Mix gently by pipetting. 
9. Incubate for 20 min on ice.
10. Add 1mL LB.
11. Shake at 30oC for 1 hour 700 rpm. 
12. Spin down (4500 RPM, 5 min), resuspend pellet in 300uL LB. 
13. Spread 50 and 150ul on a plate and incubate at 30C in Chloramphenicol plates! 
14. Keep remaining transformation mix at 4oC. 
15. Check plates next day for single colonies, and confirm by PCR/Sequencing.
TITLE: Reviving dead dry-erase markers
AUTHORS: Scott Barolo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a recipe for reviving dry-erase markers. It is based on the </span><a href="https://twitter.com/sbarolo/status/1155870573043572738" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">tweet</span></a><span> of Scott Barolo and the suggestion to add it to protocols.io is from </span><a href="https://twitter.com/neuromusic/status/1155910220855435264" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Justin Kiggins</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Tape a string to the back of the dry-erase marker as shown below.
?. Spin the marker like a lasso above your head, about 25 times.(See the video here: https://www.facebook.com/sandy.owens.54/videos/10214113211493293/.)
?. There should now be more life in the marker.
TITLE: Flow Cytometry Staining Protocol For Initial Kidney Studies
AUTHORS: Jeremy Lombardo

[DESCRIPTION]
Protocol for flow cytometry cell staining for initial kidney studies



[STEPS]
1. Cell suspensions were centrifugated (400xg, 5 min) and then stained concurrently with 5 μg/mL anti-mouse CD45-AF488 (clone 30-F11, BioLegend, San Diego, CA), 7 μg/mL EpCAM-PE (clone G8.8, BioLegend, San Diego, CA), and 5 μg/mL TER119-AF647 (clone TER-119, BioLegend, San Diego, CA) monoclonal antibodies in PBS+1% BSA (PBS+) for 30 minutes.
2. Samples were then washed twice with ~2mL PBS+ by centrifugation (400xg, 5 min).
3. Cell suspensions were then stained with 3.33 μg/mL 7-AAD viability dye (BD Biosciences, San Jose, CA) on ice for at least 10 minutes.
4. Cell suspensions were then analyzed on a Novocyte 3000 Flow Cytometer (ACEA Biosciences, San Diego, CA).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Procotol describing RNA extraction from dissected human tissue followed by reverse transcription to cDNA, PCR for detection of TMSB4X and separation of PCR products by agarose gel.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: GUV assay
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">LC3 lipidation on GUVs</div></div>

[STEPS]
?. GUV Preparation
?. Coat cleaned coverslips with 60 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore).
?. Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.
?. Spread a lipid mixture at 1 mg/ml  uniformly onto the PVA film.
?. Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.
?. Use 400 μL 400 mOsm sucrose solution for swelling for 1 h at room temperature
?. GUV Assay
?. Set up the reaction in an eight-well observation chamber (Lab Tek) at room temperature.
?. Coat the chamber with 5 mg/ml β casein for 30 min.
?. Add 10 µL GUVs to initiate the reaction.
?. Acquire time-lapse images  in multitracking mode on a Nikon A1 confocal microscope.
?. Clean the coverslips of 25 mm diameter.
?. Harvest the GUVs and use them with 12 h.
?. Wash the coated chamber three times with reaction buffer (20 mM HEPES at pH 8.0, 190 mM NaCl and 1 mM TCEP).
?. Make a 120 µL reaction mixtures with the proteins and 50 μM ATP. For E3 membrane recruitment and LC3 lipidation experiment, the final concentration of  PI3KC3-C1 complex is 50 nM, WIPI2d WT or mutant is 250 nM , E3-GFP complex is 50 nM, ATG7 is 100 nM, ATG3 is 100 nM,  and mCherry-LC3B is 500 nM. For WIPI2d membrane binding in the presence of PI3KC3-C1, the final concentration of  PI3KC3-C1 complex is 50 nM, the mCherry tagged WIPI2d WT or mutant is 400 nM. For WIPI2d membrane binding to PI(3)P GUVs, the final concentration of the mCherry tagged WIPI2d WT or mutant is 400 nM.
?. Pick random views for imaging within 5 min.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for genetic manipulation of <em>Synechocystis</em> sp. PCC 6803 using the triparental mating method described in Thiel <em>et al</em>., 1988.</p>

[BEFORE_START]
<p>Pour BG11 agar plates with 5% LB.</p>

[GUIDELINES]
<p>Thiel T, Wolk CP: <strong>Conjugal Transfer of Plasmids to Cyanobacteria</strong>. <em>Methods in Enzymology</em> 1988, <strong>153</strong>:232–243.</p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: A fast, easy, cost-free method to remove excess dye or drug from exosome solution
AUTHORS: Ioannis Isaioglou, Gloria Lopez-Madrigal, Jasmeen Merzaban

[DESCRIPTION]
This protocol details a cost-free method to remove excess dye or drug from exosome solution.

[GUIDELINES]
Tips:

Cells from step 5 should have signal detectable signal under flow cytometry and/or fluorescent microscopy assay (sponge cells). In case they do not, it is advised to increase the concentration of the dye to ensure that all isolated particles are sufficiently stained.
Cells from step 9 should have no signal (test cells). If they do, it is advised to optimize the staining conditions. For example, a reduction in the initial amount of dye can be considered. Alternatively, an increase in the number of parental cells used per washing step can also be explored with a caveat that this might reduce the number of exosomes remaining.


 


[STEPS]
SECTION: Protocol Steps
1. Collect 0.5 x 106 cells (preferably parental cells used for exosome isolation)* and wash them with
phosphate buffered saline (PBS) or alternative buffer.

 

SECTION: Protocol Steps
3. Add the mixture containing the stained exosomes** with the excess dye/drug to the cell pellet.

 

SECTION: Protocol Steps
2. Pellet down the cells (typically 350 x g, 3 min).

SECTION: Protocol Steps
4. Resuspend by gently pipetting the cells and leave the mixture shaking at 350 rpm, 15 min, 37 °C

SECTION: Protocol Steps
5. Spin down the mixture so the cells form a pellet using the same parameters as in step 2.
SECTION: Protocol Steps
6. Transfer the supernatant into a new clean tube. This supernatant should now contain the stained or loaded exosomes without any excess dye in the buffer solution used.
 

SECTION: Protocol Steps
7. Repeat steps 2 through 5: 
Pellet down the cells (typically 350 x g, 3 min).
Add the mixture containing the stained exosomes** with the excess dye/drug to the cell pellet.
Resuspend by gently pipetting the cells and leave the mixture shaking at 350 rpm, 15 min, 37 °C
Spin down the mixture so the cells form a pellet using the same parameters as in step 2.

SECTION: Protocol Steps
8. Collect the supernatant which contains the stained exosomes without the excess dye.
SECTION: Protocol Steps
9. Resuspend the pellet in PBS buffer (or alternative) and check whether the cells (test cells) were stained. This confirmation step can be verified using fluorescent microscopy or flow cytometry for fluorescent drugs or dyes.
TITLE: Interferon signalling as a potentional therapeutic target in amyotrophic lateral sclerosis and frontotemporal dementia – protocol for a systematic review and meta-analysis
AUTHORS: Fergal M Waldron, Olivia  Rifai, Jenna  Gregory

[DESCRIPTION]
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with no cure and limited treatment options. There is therefore an urgent need for effective therapeutic interventions in this disease. This protocol outlines the strategy for a systematic review and meta-analysis to identify, from in vivo animal studies, potential therapeutic interventions for ALS cases in which an interferon response is induced. Our aim is to perform a comprehensive review of the ALS literature to compile a list of (i) interventions affecting interferon signalling pathways and (ii) specific interferon signalling pathway targets that may be manipulated for therapeutic benefit in patients with ALS. We will do this by assessing the pre-clinical literature for the effects of interferon signalling pathway manipulation on the following primary and secondary outcome measures comparing models of ALS/FTD to controls; the primary outcome measure being survival, and secondary outcome measures including histological, biochemical, and behavioural metrics. We will also carry out a structured quality assessment of the literature to provide recommendations for future studies. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Nucleic acid precipitation is used to concentrate and/or purify nucleic acids. The below protocol is based on the fact that nucleic acids are less soluble in alcohol than in more polar water. Addition of salt further decreases solubility by competing for water dipoles; as does low temperature. Please see the <a href="http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids" target="_blank">OpenWetWare</a> website for more details.<br /><br /><strong>Citation</strong>: OpenWetWare contributors, 'Ethanol precipitation of nucleic acids',&nbsp;<em>OpenWetWare, ,</em>&nbsp;1 July 2012, 10:48 UTC, &lt;<a class="external free" title="http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&amp;oldid=611420" href="http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&amp;oldid=611420" target="_blank">http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&amp;oldid=611420</a>&gt; [accessed 21 January 2015]

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Q5® Site-Directed Mutagenesis (E0552)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol defines methods for the Q5® Site-Directed Mutagenesis Kit without competent cells.</div></div>

[STEPS]
?. [Exponential Amplification (PCR)]
Assemble the following reagents in a thin-walled PCR tube:  ABC1 25 μl RXNFINAL CONC.2Q5 Hot Start High-Fidelity 2X Master Mix12.5 μl1X310 μM Forward Primer1.25 μl0.5 μM410 μM Reverse Primer1.25 μl0.5 μM5Template DNA (1–25 ng/μl)1 μl1-25 ng6Nuclease-free water9.0 μl
ABC1 25 μl RXNFINAL CONC.2Q5 Hot Start High-Fidelity 2X Master Mix12.5 μl1X310 μM Forward Primer1.25 μl0.5 μM410 μM Reverse Primer1.25 μl0.5 μM5Template DNA (1–25 ng/μl)1 μl1-25 ng6Nuclease-free water9.0 μl
?. [Exponential Amplification (PCR)]
Mix reagents completely.
?. [Exponential Amplification (PCR)]
Transfer to a thermocycler and perform the following cycling conditions:Thermocycling Conditions for a Routine PCR:   ABC1STEPTEMPTIME2Initial Denaturation98°C30 seconds325 Cycles98°C10 seconds450–72°C*10–30 seconds572°C20–30 seconds/kb6Final Extension72°C2 minutes7Hold4–10°C
ABC1STEPTEMPTIME2Initial Denaturation98°C30 seconds325 Cycles98°C10 seconds450–72°C*10–30 seconds572°C20–30 seconds/kb6Final Extension72°C2 minutes7Hold4–10°C
* For a Q5-optimized annealing temperature of mutagenic primers, please use NEBaseChanger™, the online NEB primer design software. For pre-designed, back-to-back primer sets, a Ta = Tm + 3 rule can be applied, but optimization may be necessary.
?. [Kinase, Ligase & DpnI (KLD) Treatment]
Assemble the following reagents:  ABC1&nbsp;VOLUMEFINAL CONC.2PCR Product1 μl&nbsp;32X KLD Reaction Buffer5 μl1X410X KLD Enzyme Mix1 μl1X5Nuclease-free Water3 μl&nbsp;
ABC1&nbsp;VOLUMEFINAL CONC.2PCR Product1 μl&nbsp;32X KLD Reaction Buffer5 μl1X410X KLD Enzyme Mix1 μl1X5Nuclease-free Water3 μl&nbsp;
?. [Kinase, Ligase & DpnI (KLD) Treatment]
Mix well by pipetting up and down.
?. [Kinase, Ligase & DpnI (KLD) Treatment]
Incubate at  for .
0 Room temperature
?. [Transformation]
Thaw .
[aliquot of chemically competent E. coli cells]
on ice
NEB 5-alpha Competent E. coli (High Efficiency), NEB #C2987, are recommended
?. [Transformation]
Add  from the "KLD Section" above to the tube of thawed cells.
[KLD mix]
?. [Transformation]
Carefully flick the tube 4-5 times to mix. Do not vortex.
?. [Transformation]
Place the mixture  for .
on ice
?. [Transformation]
Heat shock at  for .
42 °C
?. [Transformation]
Place  for .
on ice
?. [Transformation]
Pipette  into the mixture.
[room temperature SOC]
?. [Transformation]
Incubate at  for  with shaking (250 rpm).
37 °C
?. [Transformation]
Mix the cells thoroughly by flicking the tube and inverting.
?. [Transformation]
Spread 50-100 μl onto a selection plate.
?. [Transformation]
Incubate  at .
37 °C
It may be necessary (particularly for simple substitution and deletion experiments) to make a 10- to 40-fold dilution of the transformation mix in SOC prior to plating, to avoid a lawn of colonies.
TITLE:      ELISA for quantification of IL-26 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>It is strongly associated inflammatory activity with IL-26. Th17 cells produce this interleukin. It acts on epithelial cells and intestinal epithelial cells. It induces IL-10 expression, stimulates the production of IL-1-beta, IL-6, and IL-8 and causes Th17 cell generation. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-26 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-26 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-26 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-26 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-26 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-26 standard dilutions and the human IL-26 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.