eamag/protocols-with-steps huggingface dataset:
TITLE: Human Pregnant Fallopian Tube Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC
AUTHORS: Valentina  Stanley, Scott   Lindsay-Hewett, Mana   Parast, Louise  Laurent

[DESCRIPTION]
Human pregnant fallopian tube tissue collection and storage protocol for HuBMAP's UCSD Female Reproductive TMC.

[STEPS]
SECTION: Preparation
1. Fallopian tubes should be collected into empty sterile cups right after sterilization (salpingectomy or tubal ligation). Keep left and right tubes separate.
SECTION: Preparation
2. Take a photo of the tubes prior to sampling. If pieces of the tissue were collected for clinical pathological examination, maintain the original orientation of tubes from the fimbrial (distal) end to the tubal (proximal) end after such sampling.
   

SECTION: Preserving the fimbria
4. Separate the fimbriated end from the tube (if not already separated).
SECTION: Preserving the fimbria
4.1. Divide the fimbriated end lengthwise into 2 or 3 equal sized sections (depending on size).
SECTION: Preserving the fimbria
4.2. Place one fimbriated piece into a 10% formalin-filled tube (1st priority), the other into RNAlater (2nd priority), and the third to be snap-frozen (3rd priority).
SECTION: Preserving the tube
5. Collect cross sections of the tube into 10% formalin (x1), RNAlater (x3) and to be snap-frozen (x3). Each piece should be about 5mm in length, depending on the total length of the tube. If there is an excess of tissue, the formalin piece(s) can be larger.
SECTION: Preserving the tube
5.1. Collect the formalin-preserved piece from the most distal end (closest to the fimbria).
SECTION: Preserving the tube
5.2. For the right tube, collect the RNAlater pieces from the center of the tube and the snap frozen pieces from the proximal end. T1 should be most distal, then T2, then T3 should be most proximal.
  

SECTION: Preserving the tube
5.3. For the left tube, collect the snap frozen pieces from the center of the tube and the RNAlater pieces from the proximal end. T1 should be most distal, then T2, then T3 should be most proximal.
  

SECTION: Flash-freezing
6. Drop the snap frozen labeled tubes into liquid Nitrogen. Leave for ~2-10 minutes, then store in -80C freezer.
SECTION: Processing with RNAlater
7. Place RNAlater tubes into a 4C fridge. Let these sit for 24-48 hours, then remove the RNAlater with a sterile transfer pipette and store in a -80C freezer.
SECTION: Formalin fixing
8. After one week in formalin, place each formalin-fixed piece into a labeled cassette to be processed into FFPE blocks. 
SECTION: Preparation
3. Wash the tissues in cold PBS while maintaining the original orientation.
TITLE: GWAS Fecal Collection and DNA Extraction for Downstream Sequencing Applications
AUTHORS: Sierra Simpson, Olivier George

[STEPS]
?. [Feces Collection]
Collect fresh feces – this must be directly from the animal. Dried feces from the night before, or in the cage will not yield good DNA for sequencing.Make sure the tube is labeled with an "A" for BSL, a "B" for Post-ShA, "C" for Post-LgA
?. [Feces Collection]
Place the fresh feces into a marked tube and immediately put in powdered dry ice until it can be transferred to the -80 where it will be stored. Avoid freeze thaw cycles.
?. [Feces Collection]
Take approximately two small pieces of fresh feces by gently holding the rat by the tail with the front paws on a cart and bounce the animal so the hind legs come gently off the cart – this will make the animal defecate in 30sec-1min.If the animal is not defecating, palpate the colon to determine if there are fecal pellets. If there is a fecal pellet, gently squeeze behind the pellet to release the feces from the colon.
?. [DNA Isolation for Sequencing]
Add up to 150mg fecal sample to ZR Bashing Bead Lysis Tube
?. [DNA Isolation for Sequencing]
Add 750ul Lysis Solution to the tube
?. [DNA Isolation for Sequencing]
Secure in a bead Beater fitted with 2ml holder assembly and process at max speed for 5 min.
?. [DNA Isolation for Sequencing]
Centrifuge for 10K for 1min
?. [DNA Isolation for Sequencing]
Transfer 400ul Supernatant to Zymo Spin IV Filter in a collection tube and centrifuge for 7K rpm
?. [DNA Isolation for Sequencing]
Add 1.2ml Fecal DNA Binding Buffer to the filtrate in the collection Tube
?. [DNA Isolation for Sequencing]
Transfer 800ul of mixture from step 6 to Zymo-Spin IIC column in a new collection tube and centrifuge at 10K for 1min.
?. [DNA Isolation for Sequencing]
Discard flow-though from step 7 and repeat.
?. [DNA Isolation for Sequencing]
Add 200ul DNA Pre-wash Buffer to the Zymo-Spin IIC column in a new collection tube and centrifuge at 10K for 1 min.
?. [DNA Isolation for Sequencing]
Add 500ul Fecal DNA Wash Buffer to the Zymo-spin IIC column and centrifuge at 10K for 1 min.
?. [DNA Isolation for Sequencing]
Transfer Zymo-Spin IIC column to a clean 1.7ml centrifuge tube (Denville Posi click) and add 100ul DNA Elution Buffer directly to column. Centrifuge at 10K for 30 seconds.
?. [DNA Isolation for Sequencing]
Transfer eluted DNA from step 10 to prepared Zymo-spin IV HRC filter  - into a clean 1.7ml centrifuge tube and centrifuge exactly 8,000g for 1 min.
?. [DNA Isolation for Sequencing]
The filtered DNA is now suitable for PCR and downstream applications.
TITLE: Paramecium bursaria immunofluorescence protocol
AUTHORS: David S Milner, Estelle Kilias, Thomas A. Richards

[DESCRIPTION]
This protocol has been developed for immunofluorescent localisation of host proteins in Paramecium bursaria 186b. 

[BEFORE_START]
Prepare 200 mL of PBS

  


[STEPS]
SECTION: Fixation
1. Take 6 mL of P. bursaria culture per treatment. Centrifuge 800 x g, 10 min and remove 5 mL (i.e. concentrate sample 6-fold). Aliquot 1 mL of concentrated culture into a 1.5 mL micro-centrifuge tube per treatment for downstream steps.
SECTION: Fixation
2. Centrifuge 800 x g, 10 min to pellet the cells. Remove supernatant, avoiding the cell pellet.
SECTION: Fixation
3. Re-suspend cells in 500 µL PBS containing 4% formaldehyde [375 µL PBS plus 125 µL 16% formaldehyde]. Use a fresh ampule of formaldehyde for each experiment.

Incubate for 12 min Room temperature 

 

SECTION: Fixation
4. Centrifuge 800 x g, 10 min, remove supernatant and wash once in 500 µL PBS. Repeat the centrifugation 800 x g, 10 min

SECTION: Permeabilisation
5. Re-suspend cell pellet in 500 µL PBS containing 0.05 % volume Tween 20.

Incubate for 10 min Room temperature 

  

SECTION: Permeabilisation
6. Centrifuge 800 x g, 10 min, remove supernatant and wash twice in 500 µL PBS. Repeat the centrifugation 800 x g, 10 min
SECTION: Blocking
7. Prepare PBS containing 3 % volume BSA. Filter through a 0.2 micron syringe filter prior to use. Re-suspend cells in 500 µL of this solution.

60 min Room temperature 

  

SECTION: Primary antibody
8. Add 1 µL primary antibody directly to the cells in PBS+3% BSA.

A good positive control here is 1 µL Anti-α-Tubulin antibody  

  4 °C 


 

SECTION: Secondary antibody
9. After the overnight incubation, wash three times in 500 µL PBS.

Repeat the centrifugation 800 x g, 10 min and re-suspend the cells in 500 µL PBS.

SECTION: Secondary antibody
10. Add 1 µL secondary antibody.

For rabbit primary antibodies:
  

For mouse primary antibodies (e.g. Anti-α-Tubulin antibody used above):
 

120 min Room temperature 

SECTION: Secondary antibody
12. Centrifuge 800 x g, 10 min, remove supernatant and wash twice in 500 µL PBS. Repeat the centrifugation 800 x g, 10 min and re-suspend in 20 µL of residual PBS

SECTION: Preparing slides
14. Using a wide-bore tip (i.e. a 1 mL pipette tip with the end cut-off), spot approximately 10 µL onto 
 


SECTION: Preparing slides
13. Add a drop of CITIFLUOR AF2 to the residual PBS.

  



SECTION: Secondary antibody
11. If required, add 2 µL of 10 mg/mL Hoechst 33342

 

5 min Room temperature 

SECTION: Preparing slides
15. Add a coverslip and seal with clear nail polish.
TITLE: Phylogenetic analyses of Ophiothrix (Echinodermata: Ophiuroidea)
AUTHORS: Renata Alitto, Michela Borges, Letícia Dias de Oliveira, Karin Regina Seger, Luciana Bolsoni Lourenço

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Using 16S and COI fragmens in Bayesian Inference, Maximum Parsimony, and Maximum Likelihood.</div></div>

[STEPS]
?. [Align Sequences]
All 16S and COI sequences were aligned with the MUSCLE algorithm, providing a unified matrix. All the sequences from Ophiothrix angulata (16S) and additional sequences from others Ophiotrichidae (16S, COI) available on GenBank were added to the matrix for comparison. Amphipholis squamata (Amphiuridae) was used as outgroup.
?. [Datasets]
Three datasets were considered: i) 16S, ii) COI and iii) concatenated (COI+16S). Phylogenetic reconstructions were made on the basis of two different optimality criteria, Maximum Parsimony (MP) and Bayesian Inference (BI), to assess whether there were any differences in the trees recovered with regard to the method used.
?. [Maximum Parsimony (MP)]
The MP was performed with the TNT 1.5 program. Most parsimonious trees were obtained by a heuristic search (best length was hit 100 times), using the new technology search option, which included sectorial searches, ratchet, tree drifting and tree fusing. The gaps were considered as fifth state. The cladograms had their nodes evaluated by the Bootstrap resampling test, based on 1000 pseudoreplicates using the Traditional Search.
?. [Bayesian Inference]
For BI, the best-fitting model of molecular evolution was GTR+G, chosen based on the AIC and Hierarchical Likelihood Ratio Tests according to the estimation by MR.MODELTEST v.2.3. The BI analyses used one cold and three incrementally heated Monte Carlo Markov chains (MCMC) on two simultaneous runs. The standard deviation of the split frequencies between the two runs reached a value lower than c. 0.005 at two million generations, with one tree sampled every 100th generation, each using a random tree as a starting point and a temperature parameter value of 0.2 (the default in MrBayes). The first 25% of the total sampled trees were discarded as ‘burnin’ to achieve the MCMC log-likelihoods that had become stationary and converged. The analyses resulted in similar likelihood scores, with ESS > 200, as verified using TRACER.
?. [Maximum Likelihood (ML)]
The ML was performed with the MEGA v.7.0 program using the Kimura 2-parameter model. The statistical support was obtained with a bootstrap function using 1000 replicates.
?. [Edition of phylogenetic trees]
The phylogenetic trees were visualized and edited in FIGTREE v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).
TITLE: The efficacy of telemedicine using videoconferencing system in outpatient care for cancer patients: a systematic review and meta-analysis protocol
AUTHORS: Yasuaki  Uemoto, Taro Yamanaka, Yuki Kataoka, Yoshitaka Wada, Yosuke Aoyama, Rika Kizawa, Yu Yamaguchi, Yuichiro Kikawa, Naruto Taira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Further research is needed to compare the impact of interactive Telemedicine using videoconferencing systems with usual face-to-face care in the outpatient with cancer. The aim of this study is to review systematically the efficacy of interactive telemedicine using videoconferencing systems in this setting, including feasibility, satisfaction and cost.</div><style>
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[STEPS]
TITLE:       ELISA for quantification of IL-32 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>IL-32 is a pro-inflammatory molecule. Natural killer cells and monocytes mainly produce it. IL-32 induces the synthesis of various cytokines including IL-6, and IL-1beta. It inhibits IL-15 production. </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-32 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-32 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-32 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-32 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-32 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-32 standard dilutions and the human IL-32 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Immunocytochemistry of primary neurons
AUTHORS: giulia.tombesi

[DESCRIPTION]
This protocol describes the steps to obtain a great staining of primary neurons. 

[STEPS]
SECTION: Cells Fixation
1. Wash cells with PBS1X/HBSS 
2. Fix cells with paraformaldehyde (PFA) 4% for  30 min at room temperature

SECTION: Cells permeabilization and saturation
4. Permeabilize cells with 0.3% Triton X-100 (in PBS1X) for 5 min at room temperature 

SECTION: Cells permeabilization and saturation
5. Wash cells with PBS1X for 5 min for three times at room temperature
SECTION: Cells permeabilization and saturation
6. Saturate cells in blocking solution for at least 30 min

SECTION: Cells permeabilization and saturation
7. Wash cells with PBS1X for 5 min for three times at room temperature
SECTION: Incubation with primary antibody
8. Incubate with primary antibody in working solution for 90 min at room temperature

3. Wash cells with PBS1X for 5 min for three times at room temperature    
SECTION: Incubation with primary antibody
9. Wash cells in working solution for 5 min for three times at room temperature
SECTION: Incubation with secondary antibody
10. Incubate with secondary antibody in working solution for 60 min at room temperature in the dark

SECTION: Incubation with secondary antibody
11. Wash cells in working solution for 5 min for three times at room temperature
SECTION: Incubation with secondary antibody
12. Wash cells in PBS1X for 1 min for three times at room temperature
SECTION: Mounting
13. Rinse coverslips in dH2O
SECTION: Mounting
14. Mount the coverslips on slides with mounting medium
TITLE: Cambridge Fetal Thymus Dissociation
AUTHORS: JONGEUN PARK

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is protocol for fetal thymus dissociation. (Can be applied to other fetal tissues)</div><div class = "text-block">Can be also adapted to adult thymus or lymphoid organs. (Volume of solution needs to be adjusted to take account of larger tissue size, also consider multiple rounds of digestion in this case)</div></div>

[STEPS]
TITLE: Cell viability assessment
AUTHORS: Qing Wang

[DESCRIPTION]
This is the protocol for assessing cell viability using a MTS cell proliferation assay kit.

[STEPS]
1. 1. Cells are seeded and differentiated for 4 days; assess cell viability using an MTS cell proliferation assay kit (ab197010, Abcam).
2. 2. Treat the differentiated cells with synthetic DOPA pheomelanin or synthetic DOPA eumelanin or vehicle PBS to determine the time and dose responses of the cells to the treatment.
3. 3. Remove culture medium and add a 110 µL solution containing 10 µL MTS and 100 µL medium to each well, incubate in humidified air containing 5% CO2 at 37°C for 1 hour.
4. 4. Measure absorbance at 490 nm using a microplate reader.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This method is modified from that described by Nakatani Y, Ogryzko V. Immunoaffinity purification of mammalian protein complexes. Methods Enzymol. 2003;370:430-44. Epub 2004/01/10. doi: 10.1016/S0076-6879(03)70037-8 S0076687903700378 [pii]. PubMed PMID: 14712665. It can be used to immunopurify proteins from nuclear extract through 2 sequential rounds of immunoprecipitation. Non-specific interactions are largely eliminated. This protocol is described for Flag, then HA immunoaffinity, but can be adapted for use with other epitopes.</p>

[BEFORE_START]
<p>All buffers and samples should be kept on ice or +4°C, all centrifugation steps should be performed at +4°C</p>

[GUIDELINES]
<p>This protocol is designed for use with Dignam nuclear extracts prepared from 10 plates (15cm) of HeLa S3 cells stably expressing protein-of-interest tagged Flag and HA but can be adopted for other buffers and tags</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Collecting supernatant from C. elegans culture and lyophilizing supernatant
AUTHORS: Muhammad Zaka Asif, Man Shah, Yosef Smadi

[DESCRIPTION]
This protocol describes day 5 of our workflow to grow worms in liquid culture to induce the production of natural products (in this case, 1-HP derivatives). In this protocol, we get an approximate count for the number of life-stage synchronized worms exposed to the toxin, and we collect the supernatant, which contains 1-HP and its derived metabolites. After collecting the supernatant, we freeze and lyophilize it to prepare for extraction. 

[STEPS]
1. Remove the flasks from the shaker and estimate their population by aliquoting onto glass slides as before.
2. Centrifuge at 525 RCF for 1 minute and collect the supernatant in multiple 2 mL microcentrifuge tubes.
3. Save the worm pellet at -80˚C if desired for future studies.
4. Centrifuge the microcentrifuge tubes at 20,800 RCF for 10 minutes. This will separate the bacteria from the rest of the supernatant (bacteria will settle to the bottom).
5. Combine all the supernatant remaining from the microcentrifuge tubes once the spin is completed in one 50 mL conical tube.
6. Freeze the supernatant with liquid nitrogen and lyophilize for 24-40 hrs until complete.
TITLE: Striatal injection of 6-OHDA or Saline and post-operative care (Mendonça et al 2024)
AUTHORS: Marcelo D Mendonça

[DESCRIPTION]
Striatal injection of 6-OHDA or Saline and post-operative care procedure from Mendonça et al 2024

[BEFORE_START]
Mice were kept in deep anesthesia using 1-3% isoflurane and oxygen (at 1L/min flow rate). 

[GUIDELINES]
All procedures were done in aseptic conditions. 

Experiments were done on male mice-C57BL/6 or B6.SJL-Slc6a3tm1.1(Cre)Bkmn/J (Jax labs stock #006660). 

[STEPS]
SECTION: Stereotaxic Surgery
1. Place the anesthetized mouse in the stereotaxic apparatus then stabilize the head. 
SECTION: Stereotaxic Surgery
2. Make a skin incision to expose the skull.
SECTION: Stereotaxic Surgery
3. Carefully remove any connective and muscle tissue from the exposed area. 
SECTION: Stereotaxic Surgery
4. Level the skull surface to be at less than 0.05mm by comparing the height of bregma and lambda, and also in medial-lateral directions. 
SECTION: 6-OHDA or Saline Injection
5. Find the injection coordinates and mark area for drilling: dorsolateral striatum +0.5 mm anteroposterior and ±2.5 mm lateral from bregma and 3.0 mm deep
SECTION: 6-OHDA or Saline Injection
6. Drill Burr-holes in marked areas carefully.
SECTION: 6-OHDA or Saline Injection
7. Load glass pipette into the pipette holder (Nanojet II (Drummond Scientific)) and affix to the stereotaxic frame. 
SECTION: 6-OHDA or Saline Injection
7.1. Set rate of injection to 4.6 nL per 5 seconds. 
SECTION: 6-OHDA or Saline Injection
8. Inject 2 ul of 6-OHDA or saline. 
SECTION: 6-OHDA or Saline Injection
9. After the injection finishes, leave the pipette in place for 10-15 minutes.
SECTION: Post-operative Care
10. After surgery each animal had access to up 4 mg of food pellets/day (some mice were on food restriction pre-surgery)
SECTION: Post-operative Care
11. Mice that showed weight loss were hand-fed (i.e. they were presented with the food while being held by the hands of the investigator) and DietGel Boost was placed in their boxed up to 4 days after surgery. In order to avoid competition for the food, weaker mice were placed in cages other than those containing unimpaired mice. The postoperative survival rate was 100%.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To develop broadly useful methods for the genetic manipulation of Labyrinthulomycetes, it is essential to understand the similarities and differences in regulation of gene expression among them. Toward this end we have used FIMO from the MEME suite (http://meme-suite.org/doc/fimo.html) to identify motifs similar to yeast transcription factor binding sites in each of the three available genome sequences: <em>Aplanochytrium kerguelense</em> PBS07, <em>Schizochytrium aggregatum</em> ATCC 28209, and <em>Aurantiochytrium limacinum</em> ATCC MYA-1381. We then make logograms to illustrate yeast-like GAL4 binding sites in each of the three genomes. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
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