eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol G2B study
AUTHORS: Sophie Leclercq, Camille Amadieu,  Audrey M Neyrinck,  Philippe de Timary,  Nathalie M Delzenne,  Peter Stärkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Background:The gut microbiota is a key player in the regulation of metabolism, immunity and brain functions. Our previous studies have shown that chronic alcohol abuse induces a leaky gut and alterations of the gut microbiota composition, which are correlated with the severity of psychological symptoms, suggesting the involvement of the gut-brain axis in the development of alcohol use disorder (AUD). </div><div class = "text-block">Objectives:The Gut2Brain study aims at supplementing AUD patients with prebiotics (inulin) in order to 1) modulate the gut microbiota composition and 2) to evaluate its effect on gastrointestinal tolerance, patient behaviour, biological markers and nutritional intake. </div><div class = "text-block">Design:A randomized, double-blind, placebo-controlled study included 50 AUD patients hospitalized for a 3-week detoxification program (Brussels, Belgium). Patients were assigned to the inulin (n=25) or placebo group (maltodextrin, n=25). Recruitment was completed in December 2019. </div><div class = "text-block">Methods: Change in gut microbiota composition will be assessed using microbial 16S rRNA gene sequencing. Biological (metabolites, cytokines and liver function), psychological measurements and dietary anamnesis will be performed at the beginning and the end of the detoxification program (17 days of supplementation). Gastrointestinal tolerance will be assessed throughout the follow-up.</div><div class = "text-block">The Gut2Brain study was approved by the local ethics committee (2017/04JUL/354) and is registered at www.clinicaltrials.gov as NCT03803709. </div><div class = "text-block">Keywords: Prebiotic; Inulin; Alcohol use disorder; Gut microbiota; Nutrition; Psychological symptoms; Liver function; inflammation</div></div>

[STEPS]
TITLE: Precompute Node Rankings
AUTHORS: Lillian Thistlethwaite

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how probability diffusion algorithm informs the path a network walker takes in a disease-specific network as described in Thistlethwaite et al. (2020). Briefly, using a probability diffusion walker, a network walker decides which nodes to step into, until a specified node subset has been found, or a given number of "missteps" occur.</div><div class = "text-block">Thistlethwaite L.R., Petrosyan V., Li X., Miller M.J., Elsea S.H., Milosavljevic A. (2020). CTD: an information-theoretic method to interpret multivariate perturbations in the context of graphical models with applications in metabolomics and transcriptomics. In review.</div></div>

[STEPS]
?. [wrapper_ranks.r]
This Protocol should be completed on a computing cluster, ideally. Running node ranks in serial takes a much longer amount of time, depending on the size of the network and the number of starting nodes you want to precompute node ranks for. If you launch a job for each starting node you want node ranks for and then collate those node ranks into one list object, you can process ~500 starting node node rankings in less than 15 minutes. We implement the computation of node ranks using this strategy with the following set-up:# FILE HIERARCHY: #        graphs/ folder with all bg_[model]_[type]_fold[fold].RData files in it.#        wrapper_ranks.r#        getRanks.pbs#        getRanksN.r# DIRECTIONS: Run wrapper_ranks.r on your computing cluster with for each pruned disease network. Set type="ind" #             since we want the node ranks for the pruned disease-specific network (which uses latent embedding and #             network pruning). type="noPruning" is used for the disease+reference network (no network pruning) and #             type="noLatent" is used for a disease-only network (no latent embedding or network pruning).Rscript wrapper_ranks.r [model] [fold] [type] Rscript wrapper_ranks.r "CIT" 1 "ind"Rscript wrapper_ranks.r "CIT" 2 "ind"Rscript wrapper_ranks.r "CIT" 3 "ind"Rscript wrapper_ranks.r "CIT" 4 "ind"Rscript wrapper_ranks.r "CIT" 5 "ind"Rscript wrapper_ranks.r "CIT" 6 "ind"Rscript wrapper_ranks.r "CIT" 7 "ind"Rscript wrapper_ranks.r "CIT" 8 "ind"Rscript wrapper_ranks.r "CIT" 9 "ind"# etc. for "MSUD", "MMA", "PA" and "PKU", too.
?. [getRanks.*]
TITLE: PMN- 02 - MTT Assay in Human PMN
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Published work using this protocol:</div><div class = "text-block"><span>- </span><span style = "font-weight:bold;">A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions.</span><span> Mabou Tagne A, Marino F, Legnaro M, Luini A, Pacchetti B, Cosentino M.Int J Mol Sci. 2019 Apr 13;20(8):1833. </span><span style = "font-weight:bold;">doi: 10.3390/ijms20081833</span></div></div>

[STEPS]
?. Isolate PMN according to the protocol PMN-01a for buffy coats or PMN-01b for fresh whole blood.
?. Resuspend PMN at 1x106cells/ml in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (SOLUTION- 13).
?. Add  of cell suspension per well in a 96-well round bottom plate and incubate for alone or in the presence of the test substance at  in a 5% CO2 atmosphere. Cells must be plated in duplicate for each experimental condition.
250 µl
37 °C
?. Centrifuge at  and discard the supernatant.
Centrifuge: 1400 34, 5 min
?. Resuspend cells with of MTT solution at 0.5 mg/ml, incubate for  at  in a 5% CO2 atmosphere.
200 µl
37 °C
?. Centrifuge at  and discard the supernatant.
Centrifuge: 1400 34, 5 min
?. Add  of DMSO per well, mix the plate until the formazan crystals are dissolved.
120 µl
?. Read the plate on the ELISA plate reader at 570 nm.
TITLE: Painting Neuropixels probes and other silicon probes for electrophysiological recordings
AUTHORS: Liu Liu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for consistently applying fluorescent dyes to Neuropixels probes and other types of silicon probes, which allows imaging and reconstruction of the electrode tracks in the brain in post hoc histology.</div></div>

[STEPS]
?. Secure Neuropixels probes (shown) or other silicon probes onto a micromanipulator. The side with eletrode sites should face up.
?. Secure a microscope slide onto a holder, and a clean coverslip at the end of the microscope slide. Place the coverslip attached to the microscope slide near to the Neuropixels probe (2-3 mm).
?. Using a micropipette, place 1 µL of CM-DiI solution close to the edge of the coverslip.To ensure a consistent labeling of the probe, make sure the pipette does not produce an air bubble in the drop of dye. The dye concentration should be 1 µg/µL CM-DiI in ethanol. The drop of dye will evaporate in ~30 s at room temperature, so it is important to complete step 4 quickly.
?. This step should be done under a microscope. Using the micromanipulator, dip the tip of the Neuropixels probe into the dye. Be careful not to get dye onto the electrode sites. For Neuropixels probes, the tip extends only about 150 µm from the first electrode site (see photo). The electrodes sites should be visible under the microscope. The experimenter should be familar with the setup to comfortably complete this step in considerably less than 30 s.
?. Wait until ethanol evaporates and dye dries onto the tip of the probe, around 30 seconds for ethanol-based CM-DiI.
?. Using the micromanipulator, slowly retract the probe away from the coverslip and dismount. One should be able to see dried pink dye on the tip of the probe. If not, repeat painting the probe.
TITLE: RNA Isolation from Plant Tissue Protocol 17: Invitrogen PureLink-Qiagen RNeasy Hybrid
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Patrick Edger and J. Chris Pires</div><div class = "text-block"><span>For a small number of samples the Invitrogen </span><a href="https://www.protocols.io/view/untitled-protocol-4uwgwxe/materials" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PureLink™ RNA Mini Kit</span></a><span> (Cat #12183-018A) was used to isolate total RNA while the Qiagen RNeasy </span><a href="https://www.protocols.io/view/untitled-protocol-4uwgwxe/materials" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">MinElute Cleanup Kit</span></a><span> (Cat #74204) was used to purify and concentrate total RNA. The methods followed the manufacturer’s instructions and thus they are not repeated here.</span></div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div></div>

[STEPS]
TITLE: DEVELOPMENT OF A QUESTIONNAIRE OF COMMITMENT TO NEW PHYSICAL INTERVENTIONS FOR ATHLETES: DELPHI RESEARCH
AUTHORS: Rybakova Polina Denisovna, Miroshnikov Alexander Borisovich, Shevtsov Alexey Victorovich

[DESCRIPTION]
In the context of exercise, commitment is the correct level of a person's performance of a recommended training regimen (for example, the number of training days per week), including the level at which a person follows the prescribed dosage of exercise (for example, determined by heart rate, perceived load level, or percentage of one repeated maximum). There is no questionnaire to assess exercise adherence for athletes. The consensus of experts will be used to create the questionnaire. to develop a questionnaire to assess adherence to a new training effect for athletes using the Delphi method.

[BEFORE_START]
Hypothesis: Valid and reliable assessment of exercise adherence results will allow for informed conclusions about the effects of exercise.
Round 1. According to ACCORD (items M6 and M7), we conducted a systematic literature search to create the first questionnaire on adherence to new exercise interventions for athletes [5]. Four experts reached a consensus to include 39 questions in the first questionnaire. The expert council discussed the resulting questionnaire after the first round and jointly developed a protocol for the second and third rounds. The first round protocol was registered in the OSF international database: https://doi.org/10.17605/OSF.IO/SA2E9.
Round 2. The protocol for the second and third rounds was compiled before the start of the search and did not change either during or after its completion, according to ACCORD and PRISMA-P (Preferred Reporting Items for Systematic Review and Meta Analysis Protocols (PRISMA-P))

[GUIDELINES]
The study used the Delphi method to display the current consensus among experts. The CREDES (Conducting and REporting DElphi Studies) checklist was used to ensure compliance with reporting rules and the ACCORD (ACcurate COnsensus Reporting Document) reporting guideline is dedicated to and applicable to all consensus methods used in biomedical research. The Delphi method is recommended as a reliable way to achieve consensus among experts. Delphi was planned to consist of three rounds to allow for iteration, with the ability to include additional rounds as needed.

[STEPS]
TITLE: IPTG-induced Overexpression in E. coli
AUTHORS: iGEM Dusseldorf

[STEPS]
?. [Protein Expression (optimised for inclusion bodies)]
Protein Expression (optimised for inclusion bodies)Take volume (100 ml) LB+ampicillinAdd 1 ml of the overnight cultureLet the culture grow to an OD600 of 0.6 at 37°C/180 rpmInduce with 0.5 mM IPTG (1M Stock) --> 50 µlIncubate for 2 hours at 37°C/ 180 rpmCentrifuge the culture for 10 min at 4000xg / 4°CStore cell pellet without supernatant at -80°C
?. [Protein Expression (optimised for inclusion bodies)]
Purification of inclusion bodiesThaw cell pellet on iceResuspend pellet in 1 ml water (2x50 ml pellets from 100 ml culture can be resuspended together)Transfer to 2 ml reaction tubesCentrifuge at maximum speed of a desktop centrifuge at 4°C for 15 minutesDiscard supernatantResuspend pellet in 0.5 ml BPER by vortextingRepeat vortexing every 2 minutes for 15 minutesCentrifuge at maximum speed of a desktop centrifuge at 4°C for 15 minutesStore the supernatant (BPER) in seperate reaction tubes (on ice)Dilute 1 ml BPER with 9 ml water to create 1/10 BPERWash the pellet with 1 ml 1/10 BPER (can be done by vortexing for 3x1 minute)Centrifuge at maximum speed of a desktop centrifuge at 4°C for 15 minutesStore the supernatant (1/10 BPER) in seperate reaction tubes (on ice)Resuspend pellet in 200 μl 50 mM HEPES pH 7.4, 150 mM KCl, 10% Glycerol (If you do not intend to use your protein for activity assays, water can also be used)Put samples on SDS gel or store at -20°C. On an SDS gel mix 0.5-1 µl of sample + 4 µl 5x SDS buffer and put on the gel.
TITLE: Protocols for "The female urinary microbiota in relation to the reproductive tract microbiota."
AUTHORS: Chen Chen, Lilan Hao, Weixia Wei, Fei Li, Liju Song, Xiaowei Zhang, Juanjuan Dai, Zhuye Jie, Jiandong Li, Xiaolei Song, Zirong Wang, Zhe Zhang, Liping Zeng, Hui Du, Huiru Tang, Tao Zhang, Huanming Yang, Jian Wang, Susanne Brix, Karsten Kristiansen, Xun Xu, Ruifang Wu, Huijue Jia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Human urine is traditionally considered to be sterile, and whether the urine harbours distinct microbial communities has been a matter of debate. Potential links between female urine and reproductive tract microbial communities is currently not clear. Here, we collected urine samples from 147 Chinese women of reproductive age and explored the nature of colonization by 16S rRNA gene amplicon sequencing, quantitative real-time PCR, and live bacteria culture. To demonstrate the utility of this approach, the intra-individual Spearman's correlation was used to explore the relationship between urine and multiple sites of the reproductive tract. PERMANOVA was also performed to explore potential correlations between the lifestyle and various clinical factors and urinary bacterial communities. Our data demonstrated distinct bacterial communities in urine, indicative of a non-sterile environment. </span><span style = "font-style:italic;">Streptococcus</span><span>-dominated, </span><span style = "font-style:italic;">Lactobacillus</span><span>-dominated, and diverse type were the three most common urinary bacterial community types in the cohort. Detailed comparison of the urinary microbiota with multiple sites of the female reproductive tract microbiota demonstrated that the urinary microbiota were more similar to the microbiota in the cervix and uterine cavity than to those of the vagina in the same women. Our data demonstrate the potential connectivity among microbiota in the female urogenital system and provide insight and resources for exploring diseases of the urethra and genital tract.</span></div></div>

[STEPS]
TITLE: LRRK2RCKW Widefield fluorescence microtubule binding assay
AUTHORS: David M. Snead

[DESCRIPTION]
This assay uses TMR labeled LRRK2 or LRRK1 RCKW to measure binding to microtubules in vitro.

Created by David Snead. Adapted to protocols.io by Mariusz Matyszewski.

As used in Snead, Matyszewski, Dickey et al. 2022.

[BEFORE_START]
Please take notice of the buffer preparation in section 'Materials'.
Make sure that you have labeled taxol-stabilized microtubules available. See the protocol here.

[GUIDELINES]
Similar setup as in:
 
Also see:
https://dx.doi.org/10.17504/protocols.io.ewov14qykvr2/v1

Image analysis:

Image analysis was performed with ImageJ. Average TMR-LRRK2RCKW fluorescence intensity per microtubule was calculated from a 1 pixel-wide line drawn along the long axis of the microtubule; overall average background fluorescence intensity was subtracted. These background-subtracted intensities were averaged over all microtubules per field of view, normalized by microtubule length, to yield a single data point. Eight fields of view at each concentration of LRRK2RKCW were then averaged. 

[STEPS]
SECTION: Create microscope slides:
1. Adhere Biotin-PEG-functionalized coverslips (Microsurfaces) to a microscope slide using double-sided scotch tape, creating 4 channels per slide.
SECTION: Prepare LRRK2:
6. Make sure to use TMR labelled protein. See https://dx.doi.org/10.17504/protocols.io.ewov1nq5ogr2/v1 for labeling protocol.
SECTION: Adding LRRK2 and imaging:
8. Add LRRK2RCKW sample to the channel (19 µL). Incubate for 5 min. Prepare next step while waiting.
SECTION: Adding LRRK2 and imaging:
9. Image slide. We imaged using multiple fields of view along the flow chamber with the objective in widefield illumination, with successive excitation at 488 nm (15% laser power, 100 ms exposure) and 561 nm (25% laser power, 100 ms exposure). 
Check guidelines for image analysis notes.



SECTION: Create microscope slides:
2. Add the streptavidin buffer to each channel and incubate for 3 min.
SECTION: Create microscope slides:
3. Wash twice with Wash buffer.
SECTION: Create microscope slides:
4. Add a 1:150 dilution of taxol-stabilized microtubules (19 µL per channel) and incubate for 3 min .
See https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1 for making taxol-stabilized microtubules.
SECTION: Create microscope slides:
5. Wash twice with LRRK2 buffer. Add more buffer if necessary to prevent drying out.
SECTION: Prepare LRRK2:
7. Create a working aliquot of LRRK2 (or LRRK1) in the desired concentration (ex. 25 nanomolar (nM)-50 nanomolar (nM)) in the LRRK2 buffer at Room temperature (recommended volume of 25 µL). If adding inhibitors, add them now with DMSO. Incubate for 10 min at Room temperature.
TITLE: Characterizing qPCR Assays Using Different Software Tools.
AUTHORS: Jonathan Phillips, Gregor Blaha

[DESCRIPTION]
This protocol aims to assist those new to qPCR data analysis and those who seek to escape the closed-source, proprietary software tied to their qPCR instrumentation. 
Here we describe how to assess the quality of qPCR data and qPCR assays using: 
- CFX Maestro 2.3, the software native to Bio-Rad qPCR instruments, 
- Microsoft Excel version 2404, 
- a Bayesian inference Using Gibbs Sampling (BUGS) script presented by PJ Schmidt et al., 
- the 'LOD calculator' R-script, and 
- the ‘qpcR’ R software package

[STEPS]
TITLE: Single-cell suspensions from primary human esophagus tissue
AUTHORS: Annette Hayden, Rob Walker, Jack Harrington, Lucy Kimbley, Sara Waise, Chris Hanley, Gareth Thomas, Peter Johnson, Jonathan West, Matthew Rose Zerilli, TIm Underwood

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol to dissociate fresh esophagus tissue specimens for single-cell transcriptomics.</div></div>

[STEPS]
?. Wash tissue in PBSA.
?. Mince tissue with scalpel.
?. Transfer the minced tissue to the Collagenase/DNAse solution in a 50ml Falcon.
?. Shake the suspension at 110-150 rpm for 60 mins at 37°C (with the tube on its side to increase agitation of the tissue).
?. After 15, 30, and 60 mins – pipette up and down with descending sized pipettes (25, 10 and 5 ml).
?. Pipette thoroughly.
?. Strain with a 70 um cell strainer.
?. Add 10 ml DMEM (empty) to sieve and push through with syringe plunger.
?. Keep on ice from this point.
?. Spin 1500 rpm for 5mins and remove media.
?. Resuspend pellet in RBC lysis buffer and incubate @ 4°C for 10mins.
?. Add 10 ml DMEM (empty) and pipette up and down thoroughly.
?. Pass through 40 um cell strainer – pushing through with plunger.
?. Spin at 1500 rpm for 5 mins and remove media.
?. Resuspend pellet in 1 ml of cell suspension buffer.
?. In a 0.5ml tube, add 10 ul cell solution with 10 ul trypan blue.
?. Add resultant 20 ul mix onto disposable haemocytometer (C-Chip, FR type).
?. Count the cells and record cell viability
TITLE: Data Processing  of Nano-DESI IMS data
AUTHORS: yang1832 , Julia Laskin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">Process a mass spectrometry imaging data into a final imzML file.</div></div>

[STEPS]
?. Import the raw data folder into PeakByPeak Imaging software under "Process Raw folder"
?. Set "Number of Processes" to 10.
?. Choose "File Format" as imzML.
?. Run.
TITLE: Specificity test of toehold
AUTHORS: Hung Liang Pai, Cheng-Ruei Yang

[STEPS]
?. [Preparation]
Sterilize the bench, and put on a labmat
?. [Preparation]
Thaw the reagents including 1. DNase/ RNase free water (store in -20°C) 2. solution A (store in -80°C) 3. solution B (store in -80°C) 4. RNase inhibitor (store in -20°C) 5.toehold switches with invertase DNA (store in 4°C) 6.miRNA as the trigger (store in 4°C)
on ice
?. [Prtotocols]
Gently shake and spin down the tube before adding (especially solution A and DNA plasmid)
?. [Prtotocols]
Add below reagents in to PCR tubes in order:1. ddH2O(till totally )2. solution A3. solution B4. inhibitor5. toehold switches with invertase DNA6. miRNA
5 µl
2 µl
1.5 µl
0.2 µl
50 ng
0 µl
50 µl
100 µl
150 µl
200 µl
?. [Prtotocols]
Centrifuge
Centrifuge: 4000 33
?. [Prtotocols]
Place the PCR tubes in the dry bath incubator
37 °C
?. [Prtotocols]
Put the PCR tubes   after 2 hours
on ice
?. [Prtotocols]
Measure the glucose concentrations of each PCR tubes by glucometer
?. [Prtotocols]
Put the PCR tubes in the dry bath incubator
55 °C
?. [Prtotocols]
Measure the glucose concentrations of each PCR tubes by glucometer again
TITLE: MOVAT Pentachrome Staining
AUTHORS: Charles Alpers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">The following table describes the staining color for various structures.</div><div class = "text-block">Results:    Nuclei and Elastic Fibers                 Black</div><div class = "text-block">                  Ground Substance and Mucin         Blue</div><div class = "text-block">                  Muscle                                                 Red</div><div class = "text-block">                  Collagen and Reticular Fibers          Yellow</div><div class = "text-block">                  Fibrinioid and Fibrin                           Intense Red</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakjte.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakjtn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakjtw.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakjt6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakjue.jpg" /></div></div>

[STEPS]
?. Deparaffinize and hydrate to distilled water
?. Preheat Alkaline Alcohol in 60 degree oven
?. Mordant in Bouins in microwave, covered, at Med power for 45 seconds. Let sit for 10 mins. (Do Not Dump)
?. Rinse in cold running water for 10 minutes
?. Sodium Thiosulfate for 5 minutes, wash for 5 minutes under cold running water
?. Stain in 1% Alcian Blue for 20 minutes (Do Not Dump)
?. Cold running water for 2-5 minutes
?. Place in preheated Alkaline Alcohol for 10 minutes. (in oven)
?. Cold running water for 2-5 minutes
?. Place in Movat’s Weigerts for 60 minutes (Do not Microwave)
?. Wash briefly in cold running water; rinse well in distilled water
?. Place in Crocein Scarlet/Acid Fuchsin for 1 min
?. Rinse in distilled water
?. Place in 5% Phosphotungstic Acid for 5 min
?. Transfer directly to 1% Acetic Acid for 5 minutes
?. Rinse well in distilled water
?. Dehydrate in 1 change of 95% ETOH, 2 changes of 100% ETOH 1 minute each
?. Alcoholic Saffron for 20 minutes (Do Not Dump)
?. Rinse in 2 changes of 100% ETOH for 1 minute each
?. Clear in histoclear (xylene) and mount