eamag/protocols-with-steps huggingface dataset:
TITLE: Intracellular neuromelanin quantification
AUTHORS: miquel.vila

[DESCRIPTION]
Protocol for quantifying intracellular neuromelanin 

[STEPS]
1. Scan midbrain section using the Pannoramic Midi II FL, HQ Scientific 60x
2. Acquire SNpc images at 63x with CaseViewer
3. Upload individual images at Image J software
4. Click on Adjust canvas size and adjust it at 1596x1198
5. Click on Invert image
6. With the free hand selections tool, draft a neuromelanin-pigmented neuron (excluding the nucleus) and measure the optical density (pixel brightness) and the area
7. With the free hand selections tool, draft the neuromelanin pigment of the neuron and measure the optical density (pixel brightness) and the area
8. With the free hand selections tool, draft 15-25 non-pigmented neurons (excluding the nucleus), measure the optical density and calculate the mean value
9. Normalize (i.e., substract) the values of the neuromelanin-pigmented neuron’s optical density with the mean value of the optical density of the non-pigmented neurons
10. Extra measurement: calculate the percentage of the neuronal area occupied by the pigment diving the neuromelanin pigment area by the neuron’s area
TITLE: Optimizing the electroporation parameters for Heterometopus palaeformis (strain RAJCA)
AUTHORS: Fatma  Gomaa, Johana  Rotterova, Roxanne  Beinart, Maria  Pachiadaki, Virginia  Edgcomb

[DESCRIPTION]
Cell viability after incubation with different electroporation buffers, 
Phosphate buffered saline (PBS) is used as an electroporation buffer for many eukaryotic cells. However, we observed that the PBS has a detrimental effect on the cell, causing disruption in H. palaeformis cell membrane. We then tested the tolerance of H. palaeformis to three different electroporation buffers:
A-Gene Pulser Electroporation Buffer(BIO-RAD, USA)
B-Iso-osmolar Buffer (Eppendorf, USA)
C-Hypo-osmolar Buffer (Eppendorf, USA)
Cells were incubated in each buffer independently for 10 minutes and observed for their viability under inverted microscopy. For all the three tested buffers, we noticed that once buffer was added, the cells showed instant osmic shock, include loss of motility and relative shrinking in the cell membrane (Fig. 1). After 10 minutes incubation period in each buffer, cells were transferred to their Cerophyl culture medium to recover. We observed progressive recovery of the cells. Table 1 shows the time that took the cells to completely recovered and the percentages of the recovered cells after incubation at the three different electroporation buffers. 

Table 1
     Electroporation   buffer       Duration   of recovery      Percentage   of the recovered cells        Gene   Pulser       10   minutes      >90%        Iso-osmolar         10   minutes      70%        Hypo-osmolar      2-5   minutes      70%   


 

Fig.1. Light microscopy images showing the effect of the different electroporation buffer on the H. palaeformis cell membrane; PBS has detrimental effect on the cell causing disruption of cell membrane. 
 
 
 
 
 
 
 
 
 

[STEPS]
TITLE: The relationship and interaction between stress-induced inflammation and the gut-brain (GBA) axis in the future development of post-traumatic stress disorder (PTSD) or post-traumatic stress symptoms: a systematic review
AUTHORS: jyoo, Kayla Kucela, speret, Allie Cooper, Samia Valeria Ozorio Dutra

[DESCRIPTION]
Post-traumatic stress disorder (PTSD) is a complex condition that often arises from exposure to traumatic events and affects a significant portion of the global population. Managing PTSD and understanding its multifaceted nature have become areas of heightened research and clinical interest. While there is uncertainty regarding the causes of PTSD, there is a growing recognition of the potential impact of stress-induced inflammation and alterations in the gut-brain (GBA) axis on the development and severity of PTSD symptoms. This systematic review protocol follows the PRISMA guidelines and aims to investigate the relationship between stress-induced inflammation and changes in the gut-brain axis, specifically how these factors contribute to the development of post-traumatic stress disorder (PTSD) or post-traumatic stress symptoms in adults. The study will conduct a systematic review of literature published in English from 2010 onwards, focusing on original evidence from various study designs including microbiota composition, immune responses, and gut microbiota-related markers. By conducting a comprehensive analysis of existing literature from various databases, including PubMed, CENTRAL, APA PsycINFO, CINAHL, Cochrane Library, Scopus, Sociological Abstracts, and Web of Science, this review seeks to provide valuable insights into the effects of stress-induced inflammation and gut microbiota dysbiosis on the development of PTSD symptoms, providing insights into potential mechanisms. The review is ongoing and expected to be completed by February 2024, sponsored by the College of Nursing at the University of Tennessee.

[STEPS]
TITLE: Neuropathy Phentoyping Protocols - Dosages of Tranquilizers and Anesthetics for Mice, Rats, Hamsters/Gerbils
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span style = "font-weight:bold;">Phenotyping of Rodents for the Presence of Diabetic Neuropathy </span></div><div class = "text-block">In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bim54kcn.jpg" /></div></div>

[STEPS]
TITLE: Improved protocol of ELISA for determining the serum concentration of IL-23 in humans.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A previous protocol was contaminated with IL-17. This is an improved version of an ELISA for quantification of Interleukin 23. It activates the production of IL-17A, which is a pro-inflammatory cytokine, produced by a group of T helper cell known as T- helper17 lymphocytes in response to the said IL-23 [1,2].</div><div class = "text-block">Reference</div><div class = "text-block">1. Cho ML, Kang JW, Moon YM, Nam HJ, Jhun JY, Heo SB, Jin HT, Min SY, Ju JH, Park KS, Cho YG, Yoon CH, Park SH, Sung YC, Kim HY (May 2006). Journal of Immunology. 176 (9): 5652–61. </div><div class = "text-block">2.  Nobee A, Justiz-Vaillant A, Akpaka PE, Poon-King P (2016) Levels of Interleukin 17 and 23 in Patients with Systemic Lupus Erythematosus (SLE) in Trinidad and Tobago. Immunochem Immunopathol 2: 115. doi: 10.4172/2469- 9756.1000115</div></div>

[STEPS]
?. Ninety-six well ELISA plates are coated with monoclonal anti-human antibodies to interleukin-23 (IL-23).
?. Patient serum samples are added to the plates.
?. The plate is incubate x 1.30 hour at RT.
?. The plate is washed 4 times with PBS-tween 20 buffer.
?. The wells are incubated with a biotin conjugated anti-human IL-23 for 1.30 hour at RT.
?. The plates are washed again as above.
?. To the plate a peroxidase-labeled streptavidin conjugate is added and incubated for 1 hour at RT.
?. After a further washing procedure a substrate solution reactive is added and allowed to produced a colored reaction in positive controls.
?. The level of IL-23 in the sample is proportional to the colored product developed.
?. The addition of 3M H2SO4 stops the reaction.
?. The absorbance is measured at 450 nm.
?. The IL-23 concentration can be calculated by generating an standard curve using lineal regression.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/CviJI-Purification-From-IL-3A-Virus-Infected-NC64A-er3bd8n" target="_blank">CviJI Purification From IL-3A Virus Infected NC64A Chlorella</a>.

[STEPS]
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TITLE: A Brief Look of Univariate Analysis
AUTHORS: 181830691 , FDF

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Univariate analysis mainly focuses on the description of univariate and statistical inference. It uses the simplest general form to reflect the basic information contained in a large number of sample data and describe the centralized or discrete trend in sample data. Univariate statistical inference is It is to infer the overall situation from sample data, mainly including interval estimation and statistical hypothesis testing.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Preparation of giant unilamellar vesicels (GUVs) by electroformation method has been detailed. 10 to 100 micrometer size GUVs obtained after the process. </p>

[STEPS]
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TITLE: Validation of Genotyping Method for L444P Mice Ear-Clips
AUTHORS: Revi Shahar Golan, David C

[DESCRIPTION]
Aim: the genotyping is used to identify if mice are heterozygote (hetero) or Wild-Type (WT), and the aim of the work is to validate the digestion method, and PCR program, the PCR primers, and the interpretation of the results. 

General notes: There are two sets of primers – Neo primers to distinguish between WT and hetero samples, and L444P primers to detect L444P protein. Negative and positive samples are used to verify the digestion and PCR, by gel electrophoresis. Sequencing is used to verify the interpretation of the gel electrophoresis.

[GUIDELINES]
The sequence: (ACCESSION NC_000069) we look at GACTTGGAAACAG (about 230 to 240 on a sequencing histogram): hetero/WT are shown below, with the yellow highlighted part can either be ttg/ccg (L/P) for hetero (+/Δ), or ttg (L) for wt (+/+).
  

[STEPS]
SECTION: Digestion
1. Prepare Digestion mix: 15 µL (Roche, cat 10241100) per 1000 µL (Viagen, cat 402-E).

SECTION: Digestion
2. Add 50 µL to each ear clip sample.

SECTION: PCR
7. 
Prepare PCR master mix (MM) as below (per sample):

2.5 µL (VWR-Peqlab, cat 01-1020) 
0.25 µL (VWR-Peqlab, cat 01-1020) 
0.5 µL (10pmol/ µl)
0.5 µL (10pmol/ µl) 
0.5 µL (40mM; VWR, cat 5100850-0500)
19.25 µL (Promega, cat P1193)
 

SECTION: Gel electrophoresis
11. Run PCR products on a pre-cast 2% SYBR-safe E-GEL (ThermoFisher, cat G521802), for 30 min, along with a 100bp DNA ladder (NEB, N3231), and a negative control (no DNA, only MM). 
 

 

 

 

SECTION: Digestion
3. Heat for 55 °C, 300 min.

SECTION: Digestion
4. Heat 99 °C for 10 min.

SECTION: Digestion
5. Cool to 10 °C.

SECTION: Digestion
6. Samples can be stored in -20 °C until use.

SECTION: PCR
8. For each sample, add 23.5 µL and 1.5 µL.

SECTION: PCR
9. PCR program:
 

SECTION: PCR
10. PCR product can be kept in 4 °C for the short term, or at -20 °C.

SECTION: Routine check of ear clips:
12. Run digested ear clips with NEO primers in the first instance. Then choose one of three options: 
If the ratio wt:hetero is 1:3, there is no need for further tests. Wt animals to be terminated. 
If there are more wt than the 1:3 ratio, this suggested poor DNA for the samples that were not amplified, so need to amplify with L444P primers the samples that did not work, and sequence them. 
If there are much more hetero than the 1:3 ratio, need to sequence all the samples.
TITLE: Chlamydia pneumoniae-Induced Neuroinflammation Cell Model Using Lyophilized Cell-Free Supernatant 
AUTHORS: Elif  Kaya Tilki

[DESCRIPTION]

Chlamydia pneumoniae (Cpn) is a gram-negative intracellular pathogen that causes a variety of pulmonary diseases, and there is growing evidence that it may play a role in Alzheimer's disease (AD) pathogenesis. Cpn can interact functionally with host histones, altering the host's epigenetic regulatory system by introducing bacterial products into the host tissue and inducing a persistent inflammatory response. Because Cpn is difficult to propagate, isolate, and detect, a modified LPS-like neuroinflammation model was established using lyophilized cell free supernatant (CFS) obtained from infected cell cultures, and the effects of CFS were compared to LPS.

[STEPS]
1. HEp-2 cells were used as a host to inoculate Chlamydia pneumoniae. A 6-well plate of 1X106 HEp-2 cells was seeded 48 hours prior to inoculation with Chlamydia pneumoniae (ATCC 53592).
2. The suspension of elementary bodies diluted in infection medium was added directly to wells.
3. The mixture was centrifuged at 1500 × g for 1 h.
4. The plate was incubated for 1 h at 37 °C in the presence of 5% CO2.
5. Current medium was discarded.
6. Cells were washed with 300 μL Hanks Balanced Salt Solution.
7. 500 μL fresh medium was added to the wells.
8. The plate was incubated for 72 hours.
9. The inclusion bodies were confirmed using the Pathfinder Chlamydia Culture Confirmation System (Cat. No. 30701, Bio-Rad, Germany) according to the kit manual.
10. The cells were imaged using the Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, USA).
11. The number of inclusion-forming units per milliliter (IFU/ml) in HEp-2 cells was used to determine the infectivity titers of chlamydial stocks and 1x106 HEp-2 monolayers in 6-well plate were contaminated with Chlamydia pneumoniae suspended in inoculating media at 1 multiplicity of infection (MOI) ratio.
12. Cell-free supernatant was collected from the wells, lyophilized and stored in aliquots at -80 °C.
13. The lyophilized cell-free supernatant was weighed to prepare a main stock in the desired ratio and used to trigger the inflammatory response.
TITLE: Extraction of DNA from endocervical samples using QIAamp DNA mini kit
AUTHORS: emmanuelgomseu EBDG EBDG Boris Emmanuel DJOUMSIE GOMSEU

[DESCRIPTION]

DNA was extracted from endocervical samples using the QIAamp mini kit according to the manufacturer’s instructions.
1 Centrifuge tubes containing the endocervical specimens at 15,000 rpm for 10 min. 
2 Remove supernatant and preserve the pellet; 
3 Add  180 µL of the ATL buffer and homogenize with the help of a vortex mixer.
4 Add 20 µL Proteinase K to the homogenized mixture and mixe by vortexing 
5 Incubate the solution in a dry bath at  56 °C  for 180 min  while homogenizing 2 to 3 times during incubation. 

6 Add 200 μL AL of buffer and homogenize again using a vortex for  15 s 

7 Incubate the solution in a dry bath at70 °C   for 10 min . 
8 Add 200 µL  of ethanol and homogenize. 
9 Then transfer the mixture carefully into a QIAamp mini spin column and centrifuge at 8,000 rpm for 1 min .
10 Discard filtrate and place the minicolumns were placed QIAamp mini spin column in new 2 mL tubes
11 Add of 500 µL  buffer AW1, centrifuged at 8,000 rpm for 1 min  and discard the filtrate. 
12 Add of 500 µL  buffer AW2 was added to minicolumns.
13 Centrifuge at 14,000 rpm for 3 minutes and discard the filtrate. 
14 Centrifuge the spin column with the collection tube again at 14,000rpm for 1 min 
15 Place minicolumns in 1.5 mL micro-centrifuge tubes and add 50 µL  of buffer AE. 
16 Incubate at room temperature for 5 minutes and centrifuge the mixture at 10,000 rpm for 3 min .
17 Discard the QIAamp mini spin column and store micro-centrifuge at -20 °C for subsequent molecular analysis. 

















[STEPS]
TITLE: Spot detection on structured background protocol
AUTHORS: Bin Fu

[DESCRIPTION]
This protocol details the steps for using the spot detection code

[STEPS]
1. Put sub-diffraction beads images in the folder ‘area_threshold’ and run determineAreaThres.m to find the maximum area in pixel for a diffraction-limited object.
2. Put negative control images in the folder ‘negative_control’ and run determineRad.m to determined the steepness and integrated gradient used in the study.
3. Put images in ‘image’ folder and run detection.m for diffraction-limited puncta detection
TITLE: Yale Murine TMC -  Frozen Tissue Sectioning Protocol for Spatial Transcriptomics (DBiTSeq) and Immunofluorescence  
AUTHORS: Yun-Hee Youm, Yufeng Liu, Bo Tao, Vishwa Deep Dixit, Rong Fan

[DESCRIPTION]
Protocol for frozen tissue sectioning for use in DBiTSeq and immunofluorescence staining

[STEPS]
SECTION: Tissue Embedding
1. Embed tissue according to this protocol: dx.doi.org/10.17504/protocols.io.kqdg3x8keg25/v1
SECTION: Cryosectioning
2. Spray the workplace and tools (blades, tweezers, brushes, etc) with RNAseZap to decontaminate the environment.  Spray 100% ethanol onto the cryostat to remove any residue of RNAseZap or reagent residue.  Set the cryostat to -20°C.  Prepare dry ice in a foam box.  Place the empty clean slide box in the dry ice.
SECTION: Cryosectioning
3. Remove tissue blocks from the -80°C freezer/dry ice and allow them to warm in cyrostat to sectioning temperature (-20°C) for 20 minutes.  Locate the region of interest in the tissue block.
SECTION: Cryosectioning
4. Trim the block if needed.  Collect the defect tissue slides on a regular glass slide to test for RIN number.  Usually, 3-4 sections of a 1 cm x 1 cm tissue on a glass slide is good for a RIN test.
SECTION: Cryosectioning
6. One section per slide needs to be put onto the center of the Ultra clean poly-L-lysine glass slide (see picture below).  The glass slides are at room temperature.  Label the bottom side of the slide for sample name with a marker.  Make sure the top side of the slide is free of dust or any labeling.  Use Dustoff gas to blow off the surface.  
   
SECTION: Cryosectioning
7. Once the tissue slice is picked up onto the glass slide, allow 30 seconds to 1 min at room temperature before transferring to a slide box in dry ice. 
SECTION: Cryosectioning
8. Save the slide box in a sealed plastic bag at -80° C before use.  The bag helps prevent water condensation on the slides.
SECTION: Cryosectioning
9. For slides used for H&E, thaw them at room temperature for 10 min, then dry them with compressed air for at least 5 min.  Make sure no moisture is observed on the slide.  Dip the slides in 4% paraformaldehyde at room temperature for 15 min (alternative: 30 min in pre-chilled methanol at -20° C).  Wash with deionized water 3 times.  Air blow dry the slides or leave them at room temperature overnight before doing H&E staining.
SECTION: Cryosectioning
5. Adjust cyrostat thickness to 7-10 μm.  For spatial transcriptomes, 7-10 μm is the best thickness, but generally 8-13 μm works as well.
TITLE: Identification of a Plasmid: Transformation
AUTHORS: SGD

[DESCRIPTION]
Protocol for the identification of a plasmid by using transformation of E. Coli

[STEPS]
SECTION: Transformation
1. Transfer 1 mL E. Coli to each of two 1.5 mL Eppendorf tube

SECTION: Transformation
2. Centrifuge at full speed for 30 s, then remove the supernatant

SECTION: Transformation
3. Add 450 µL of TFB1 to each tube, then resuspend gently by pipetting up and down (do not vortex)
Keep everything on ice
Incubate on ice for 10 min

SECTION: Transformation
4. Pellet the bacteria by centrifugation at full speed for 30 s
Remove the supernatant
Resuspend the pellets in 100 µL of TFB2
Incubate on on icefor 10 min

SECTION: Transformation
5. Add 100 ng of plasmid DNA to the competent cells, and mix gently

SECTION: Transformation
6. Heat shock the cells by transferring directly from the ice to the 37 °C hot block for 5 min
Then incubate on ice for 5 min, and add 1 mL LB broth to each tube

SECTION: Transformation
7. Incubate again at 37 °C, for 30 min, and allow the cells to recover

SECTION: Transformation
8. Pellet the bacteria again by centrifuging at full speed for 30 s
Discard the supernatant
SECTION: Transformation
9. Resuspend the pellet in 400 µL of LB broth
SECTION: Transformation
10. Spread 100 µL of cells per plate, onto the appropriate antibiotic plates

SECTION: Transformation
11. Incubate the plates 30 s at 37 °C
TITLE: Supporting information 1 (Yuzhalin et al. 2019)
AUTHORS: Yuzhalin AE, Lim SY, Gordon-Weeks AN, Roman Fischer, Kessler BM, Dihua Yu, Muschel RJ

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Supporting Information 1. Raw results obtained from quantitative label-free proteomics analysis of decellularized and enriched tissues of the mouse intact liver (control) and MC38 mouse liver metastasis (tumor).</div></div>

[STEPS]
?.
TITLE: 3D Reconstruction of Neurons in Vaa3D v 3
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to generate accurate digital representations of neuron morphologies from a variety of brain regions and species. Each reconstruction captures the positions and thicknesses of the soma, dendrites and axon of a biocytin-filled cell within a slice of brain tissue. To generate the reconstruction we use an image stack containing ~200-700 serial 2D images that captures the full extent of the cell within the slice. We use the Vaa3D (Terafly) program with a Kazom’s Mozak user interface (Mozak for short) to visualize the 2D images in 3D. Once the stack is loaded in Mozak, our reconstruction is generated by placing nodes in 3D space. The placement of these nodes is dependent on the signal in the images. Our final output, an SWC text file (.swc format), contains many thousand rows. Each row contains a node ID, an x, y, z coordinate, radius value, neurite type, and parent node ID.  After tracing is complete, we preform post processing to provide radius values, check for errors and consistency then the SWCs are uploaded into our Laboratory Information Management System.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]