eamag/protocols-with-steps huggingface dataset:
TITLE: Primary cortical mixed culture
AUTHORS: Xiqun Chen, Qing Ye

[DESCRIPTION]
To obtain cortical culture, pregnant mice were anesthetized, embryos were dissected, and cortex was collected in PBS. Tissues were incubated in 0.25% trypsin–EDTA at 37°C for 15 min. Trypsinized tissue was transferred into a high-glucose DMEM/F12 medium supplemented with 10% FBS. After centrifugation (1500rpm, 5 min), the pellet was resuspended. Cells were plated onto poly(L-lysine)-coated 24-well plates at 106cells per well and cultured in NB-A.

[STEPS]
1. For primary cortical mixed culture - Use C57BL/6J mice at embryonic day 17 
2. Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex. 
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
3. Collect the cortices in PBS  in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) on ice 5 mL ) on ice 

4. Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37 °C for 15 min Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times
5. Centrifuge the dissociated cortices (1500 rpm, 5 min) and resuspend the pellet in 10ml 
  
 medium supplemented with 10% 
 

6. Seed the cells onto poly(L-lysine)-coated 24-well plates at a cell density of 1x106 cells/well containing specialized   (NB-A) supplemented with 10%  

 
7. After 5 days of culture, these cells are ready for further experiment
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Checklist:</p>
<p> </p>
<p>1. FASTA file containing the proteins to analyze.</p>
<p>2. FASTX toolkit installed</p>
<p>3. phobius installed</p>
<p>4. pandas installed in python</p>

[GUIDELINES]
<p>This protocol was executed via ssh on a Ubuntu machine using the command line.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Liver Tissue dissociation of single-cell RNA-seq (10x Single-cell RNA-seq)

[STEPS]
SECTION: Buffers
?. 
SECTION: Enrichment of Hepatocytes
?. 
SECTION: Dissociation
?. 
SECTION: Dead Cell Removal
?. 
SECTION: MACS depletion of CD45 positive cells
?.
TITLE: Photographing Agave for 3D reconstruction using Structure From Motion 
AUTHORS: David Lebauer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was used in 2014 to capture 3D models of plant canopies. The technology has certainly advanced since this time! </div><div class = "text-block"><span>Our goal was to obtain a representation of a plant as a triangluar mesh that could be used for phenotyping, biomass estimation, and simulation of light interception. All of the data collected in this effort is available here: </span><a href="https://uofi.box.com/v/ebi3Dcanopy" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://uofi.box.com/v/ebi3Dcanopy</span></a><span>. This folder contains images of five agave plants and a handful of 3D reconstructions </span><a href="https://uofi.box.com/v/dec2014agave" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://uofi.box.com/v/dec2014agave</span></a><span>.   </span></div><div class = "text-block">The general approach is to take photographs of a plant from all angles. This could be as much as sixty photographs, in three rounds of twenty - to give a conservatively high number. This should be done on a day with low wind and difuse light (the sun should not be visible - either fully cloudy or when sun is below the horizon).</div><div class = "text-block"><span>Here we describe how to take pictures of an </span><span style = "font-style:italic;">Agave </span><span>plant to generate photos that can be reconstructed into a 3D point cloud or triangular mesh representation of the plant using Structure From Motion  software such as Agisoft Metashape.</span></div></div>

[STEPS]
?. place sheets of newspaper, sheets, or other material on the ground around the base of the plant, and in the background. It is most important to obscure other plants.
?. take a photograph from eye level in which the plant takes up most of the frame.
?. move approximately 20 degrees ( two steps to the side) around the plant and take another photograph.
?. repeat 1-2 18 times until you have gone in a circle.
?. repeat 1-3, but taking from the ground looking up at the plant
?. repeat 1-3 but from an angle looking down at the plant
?. download the images to a computer and share using Box. Here is a collection of images from a photo shoot in December 2014 https://uofi.box.com/v/dec2014agave
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Background: The two most common surgical approaches to total hip arthroplasty are the posterior approach and lateral approach. The surgical approach may influence cup positioning and restoration of the offset, which may affect the biomechanical properties of the hip joint.<br />The primary aim was to compare cup position between posterior approach and lateral approach. Secondary aims were to compare femoral offset, abductor moment arm and leg length discrepancy between the two approaches.<br />Methods: Eighty patients with primary hip osteoarthritis were included in a randomized controlled trial and assigned to total hip arthroplasty using posterior approach or lateral approach. Postoperative radiographs from 38 patients in each group were included in this study for measurement of cup anteversion and inclination. Femoral offset, cup offset, total offset, abductor moment arm and leg length discrepancy were measured on preoperative and postoperative radiographs in 28 patients in each group.</p>

[STEPS]
TITLE: Self-Perception: A view into the mind of Self-Imaging
AUTHORS: Hunter Duval

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">When a person looks into a mirror, three reflections appear the reflection that only they see, the reflection that everyone else sees, and the reflection that is truly there. This is due to a mental phenomenon called self-perception, or the way a person views themself, whether it be true or false. A study was conducted by Hunter Duval, to determine the relationship between demographic information (i.e. Age, Race, Etc) and Self-Perception of individuals. The website, SurveyMonkey was used. 71 individuals from the social media platform, Reddit, and a local high school were interviewed regarding their Gender, Sexual Orientation, Race, and Perception. The study concluded that 51.62% of males, 64.86% of females, and 33.33% of other genders perceive themselves as unattractive, with females having the majority. The study also found that individuals who are part of the LGBT minority have a higher chance of perceiving themselves as unattractive than that of heterosexual, with Pansexual taking the majority. According to this data, oppressed populations and minorities seem to have a higher chance of feeling unattractive. This is likely because they are, as aforementioned, oppressed. Oppressed populations, such as those in the LGBT community, are forced to look down upon themselves by other peoples, thus causing their self-image to downgrade. Overall, minority populations are more likely to have a poorer self image than that of the majority.</div></div>

[STEPS]
?. Choose Population — Be sure to choose an unbiased population, meaning do not choose a population that is known for being over or under-confident.
?. Set Up Questions — What are the subjects going to be asked? How will these questions pertain to the experiment?
?. Collect Data — Ask the population the selected questions and record their responses; OR have the population take a survey with the selected questions.
?. Analyse Data — What do the numbers mean? How much of a certain sub-population perceive themselves as attractive?
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines the beta-diversity analysis that we performed on the whole metagenome samples. We measured the signifiance of the dissimilarity between samples that were grouped by biological occlusion status, microenvironment (sebaceous, moist, etc), and sampling time point. Here we also describe our intrapersonal vs interpersonal dissimilarity measurements. Based on methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.<br /><br /><br />

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Simple Step toy Increase Gibson Assembly Efficiency 
AUTHORS: Eric Danner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Gibson assembly solutoin is extremely toxic to some bacteria cells. </div><div class = "text-block">Doing a solution change after assembly and before transforming the bacteria will significantly improve bacteria survival. This increases 10-100x the amount of colonies.</div></div>

[STEPS]
?. [Do Normal Assembly]
Do a normal Gibson assembly. Incubate in the PCR machine 60 min @ 50C
?. [Do Normal Assembly]
Remove the assemblies. Do a PCR quickchange/ gel extract column. This will remove the Gibson Assembly solution but keep the 100ng (or however much) of DNA.Elute in 15u -20ull TE or water
?. [Do Normal Assembly]
Take the 15-20ul assembled plasmid and heat shock your bacteria. 30 second heat shock. 2 min ice. Add 200ul media and shake for 45 min. Carb can be plated immediatly. Plate
TITLE: HuBMAP | GE/UPitt Cell DIVE™ Modality Overview
AUTHORS: Liz McDonough

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an overview of all protocols currently in use for the GE/UPitt Cell DIVE collaboration for the Human BioMolecular Atlas Program (HuBMAP). It includes links to each of the individual protocols that make up this project workflow.</div></div>

[STEPS]
?. Confirm donor acceptance criteria for inclusion.Donor Acceptance Criteria for GE/UPitt HuBMAP Inclusion
?. Prepare paraffin blocks and FFPE sections from tissue samples.HuBMAP | Formalin Fixation and Paraffin Embedding of Tissue SamplesHuBMAP | Sectioning of FFPE Specimens
?. Deparaffinize and rehydrate slides.Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval
?. Characterize antibodies (primary/secondary, direct conjugates, and zenon labelled antibodies) and determine any antigen effects from the Cell DIVE dye inactivation process.Cell DIVE™ Platform | Antibody Characterization for MultiplexingCell DIVE™ Platform | Antibody Staining & Imaging
?. Prepare direct conjugates for study.Cell DIVE™ Platform | Antibody Purification ChemistryCell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation
?. Perform Cell DIVE™ multiplexed data acquisition on the final cohort.
Staining is done using the Leica Bond MAX and images are acquired on the IN Cell Analyzer 2200.
TITLE: Assay for Dual Rab GTPase binding to the LRRK2 Armadillo Domain
AUTHORS: Claire Y Chiang, Suzanne R Pfeffer

[DESCRIPTION]
The LRRK2 Armadillo domain contains multiple Rab GTPase binding sites.  To show that the sites can be occupied simultaneously, we use this assay.  The idea is to immobilize Rab8A, bind Armadillo domain, and test if phosphoRab10 can bind to Rab8A-immobilized Armadillo domain.

[STEPS]
SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
1. Phosphorylate His-Rab10 Q68L 1-181 with His-MST3 kinase at a molar ratio of 1:3 (kinase:substrate) at 30 °C 120 min in reaction buffer.  See below for details.
 


SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
2. Pellet 50 µL glutathione agarose slurry at 3000 rpm, 5 min, 4 °C.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
3. Add GST-Rab8A Q67L to glutathione beads to achieve a concentration of 6 micromolar (µM) in a total volume of 50 µL reaction buffer.  Incubate at 23 Room temperature for 30 min on a rotator.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
4. Pellet beads by spinning at 3200 x g, 30 s and discard supernatant.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
5. Add His-LRRK2 Armadillo domain 1-552 (10 micromolar (µM) final in 50 µL) or buffer alone and incubate at Room temperature for 30 min on a rotator.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
6. Pellet beads by spinning at 3200 x g, 30 s and discard supernatant.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
7. Add phosphorylated His-Rab10 Q68L 1-181 (4 micromolar (µM) in 50 µL) and incubate at Room temperature for 30 min on a rotator.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
8. Wash beads 2X with 500 µL reaction buffer.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
9. Elute protein from beads using 50 µL elution buffer (reaction buffer + 50 millimolar (mM) reduced glutathione).

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
10. Pellet beads by spinning at 3200 x g, 30 s and collect supernatant.

SECTION: Dual Rab GTPase binding to LRRK2 Armadillo Domain
11. Analyze eluate by SDS-PAGE and immunoblot for phosphoRab10; image blots with Li-COR, and quantify bands using ImageJ (see below for details).
TITLE: Total Lipid Extraction from Baker&#39;s yeast (Saccharomyces cerevisiae)
AUTHORS: Israel Olayide, Monica  Rieth

[DESCRIPTION]
This protocol outlines a method for extracting total lipids from Baker's yeast, Saccharomyces cerevisiae.  It has been adapted from Roy et al., J. Lipid Res. 2018. doi: 10.1194/jlr.M088559.

[GUIDELINES]
All culture ODs should be normalized for comparative analyses prior to the extraction.  Weighed Eppendorf tubes to be used before getting started.
Extracted lipids can be stored at -20 degC

[STEPS]
SECTION: Day 1-3
1. Sterilize the inoculating loop by dipping it in the ethanol and heating it for 30 second in the flame
SECTION: Day 1-3
1.1. Allow loop to cool the loop at least for 10 seconds

SECTION: Day 4
4. Inoculate 5 mL YPD with yeast colony from plate 
SECTION: Day 4
5. Grow overnight at 30°C with shaking at 250 rpm 
SECTION: Day 5
6. Check OD600 at UV-Vis spectrophotometer
SECTION: Day 5
12. Vortex vigorously for 5 mins, and centrifuge at 150 x g for 5 mins at room temperature.
SECTION: Day 5
13. After centrifugation, a two-phase system is obtained: aqueous top phase and organic bottom phase which contains the lipids.
SECTION: Day 5
14. Carefully remove the top aqueous layer and the middle insoluble layers (precipitated proteins).
SECTION: Day 5
15. The organic bottom layer is dried using a speed vacuum or dried under a steady stream of nitrogen.
SECTION: Day 5
16. Record the weight of the dried sample by pre-weighing an Eppendorf tube or equivalent before or after drying the lipid.
0.20. Incubate plate at 30°C until colonies 1-2 mm in diameter have formed (2-3 days) 
3. Allow plate to incubate for 2-3 days at 25-30 °C until colonies are 1-2mm in diameter

SECTION: Day 5
7. Harvest the whole cells when OD600 is 1.5
Centrifuge at 1000 x g for 2 minutes or until clear to pellet the yeast
SECTION: Day 5
8. Pour off the supernatant without disturbing cell pellet
SECTION: Day 5
9. Wash the cells three times by resuspending pellets in 1ml PBS, centrifuge for at 500 x g for 3-5 mins.
SECTION: Day 5
10. After washing three times with PBS, resuspend in 1ml PBS and transfer this mixture to a 15 ml conical centrifuge tube.
SECTION: Day 5
11. Add 3.75 mL of chloroform/methanol (1:2) to the 1 mL of resuspended cells
Add 1.25 mL chloroform and 1.25 mL sterile water subsequently.
2. Streak plate from prepared glycerol stock (-80 °C) and spread on YPD agar plate
TITLE: Imaging- Bright field
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Protocol for imaging using Hamamatsu NanoZoomer slide scanner. Sections for analysis should be mounted on slides, stained for appropriate markers, and coverslipped. This protocol is using a Carl Zeiss LSM 880 confocal microcope.

[STEPS]
1. Image slides at 40x magnification.
2. Extract individual  sections using the NDP.view software (https://www.hamamatsu.com/eu/en/product/life-science-and-medical-systems/digital-slide-scanner/U12388-01.html).
3. Save images as tiff files.
4. Open images from same structures as one single stack using the merge function.
5. Using the crop, rotate, and move functions align all
images from the same stack as best as possible. Note: Using 2-3 specific
anatomy points to align the structures make the process easier.
6. Crop the entire stack at the same dimensions.
7. Save each image as a new tiff file (it is recommended
to save it in a new folder).
8. Open the imageJ macro provided on Zenodo: https://doi.org/10.5281/zenodo.10822458. 
Note: A README is provided at the beginning of the macro in order to convert all images in a 0-255 bit image.
9. Save the images as a new tiff file (it is recommended to save it
in a new folder).
10. Open matlab and run for each structure the protocol provided on the Zenodo: https://doi.org/10.5281/zenodo.10822458. 
Note: Follow the information provided on the code to align images.
TITLE: Euplotes crassus GFP artificial nanochromosome sequence
AUTHORS: Angela Piersanti, Rachele Cesaroni

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Euplotes crassus </span><span>GFP artificial nanochromosome </span></div></div>

[STEPS]
?. CCCCAAAACCCCAAAACCCCAAAACCCCTGAAGGAAGGCCGTCAAGGCCGCATATTAACTAGGTGAAATGTTGGTTATAAATATTTAAAAATGTCTGGAGGAGAAGAACTTTTCGCTGGAATTGTTCCAGTTCTTATTGAACTTGATGGAGATGTTCATGGACATAAGTTCTCTGTTAGAGGAGAAGGAGAAGGAGATGCTGATTATGGAAAGCTTGAAATTAAGTTCATTTGTACTACTGGAAAGCTTCCAGTTCCATGGCCAACTCTTGTTACTACTCTTTGTTATGGAATTCAATGTTTCGCTAGATATCCAGAACATATGAAGATGAATGATTTCTTCAAGTCTGCTATGCCAGAAGGATATATTCAAGAAAGAACTATTCAATTCCAAGATGATGGAAAGTATAAGACTAGAGGAGAAGTTAAGTTCGAAGGAGATACTCTTGTTAATAGAATTGAACTTAAGGGAAAGGATTTCAAGGAAGATGGAAATATTCTTGGACATAAGCTTGAATATTCTTTCAATTCTCATAATGTTTATATTAGACCAGATAAGGCTAATAATGGACTTGAAGCTAATTTCAAGACTAGACATAATATTGAAGGAGGAGGAGTTCAACTTGCTGATCATTATCAAACTAATGTTCCACTTGGAGATGGACCAGTTCTTATTCCAATTAATCATTATCTTTCTACTCAAACTAAGATTTCTAAGGATAGAAATGAAGCTAGAGATCATATGGTTCTTCTTGAATCTTTCTCTGCTTGTTGTCATACTCATGGAATGGATGAACTTTATAGATAAAGGAACTCAAATTTTTGCTAAAGATTTGTGGGAGAATCTTAGTCAAATTGGCAGAATTATTCAACTGTGTATTCTAATAAACTGGGCCTCATGGGCCTTCCTTTCGGGGTTTTGGGGTTTTGGGGTTTTGGGG
TITLE: TS Procure 812 - H&amp;E stained section under glass coverslip on glass slide (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
The method is used to process H&E stained slides through to Procure 812 resin blocks. 

[GUIDELINES]
Sample is not optimal for EM, best to dissect and use only the outer 2mm of tissue mass. Sample is usually derived from histology.

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR & STEPS:




OPERATOR & STEPS:
SECTION: Remove Coverslip
3. Remove coverslip by soaking in    minimum   , check to see if coverslip has slide off, if not soak another   .
SECTION: Conventional
7. 2.5 % volume   in 0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 5 min  

SECTION: Conventional
8. 0.1 Molarity (M) buffer 5 min 

SECTION: Conventional
9. 1 % volume   in buffer 30 min 

SECTION: Conventional
10. 70 % volume   for at least 5 min  or overnight

SECTION: Conventional
11. 95 % volume    5 min 

SECTION: Conventional
12. 100 % volume    5 min 

SECTION: Conventional
13. 100 % volume    dried with molecular sieve 5 min 


SECTION: Conventional
14. 50 % volume   50 % volume   10 min 

SECTION: Conventional
15. 100 % volume   10 min 

SECTION: Conventional
16. 50 % volume   50 % volume   20 min 


SECTION: Conventional
17. 100 % volume   20 min 

SECTION: Conventional
18. 100 % volume    60 min 

SECTION: Conventional
19. Embed & polymerise overnight in oven at 60 °C 

SECTION: Rehydration
4. 100 % volume   5 min 

SECTION: Rehydration
5. 95 % volume   5 min 

SECTION: Rehydration
6. 70 % volume   5 min 

SECTION: Prepare slide
2. Using diamond pen etch a circle on the back of the slide to indicate where the BEEM capsule should be placed during embedding.
SECTION: To remove the block from the glass slide
20. Place slide into boiling water on hot plate setting 3 for 9 min .Remove from water, gloves on, use tissues to hold slide down, angle single edged razor blade at 45o underneath tap washer to make a start to removal , then manually break BEEM capsule off slide.
TITLE: 3D Immunostaining for CLARITY-processed samples
AUTHORS: Seth Currlin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a guide for immunostaining CLARITY-processed samples. </span><a href="https://dx.doi.org/10.17504/protocols.io.8jihuke" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.8jihuke</span></a></div><div class = "text-block"><span>These steps are meant to be a guide for immunostaining large samples and should be optimized to suit your particular tissues and reagents. Large tissue volumes and dense tissue types will require longer incubation and wash times. The parameters suggested below are for a piece of human thymus tissue approximately 5 mm</span><span style = "vertical-align:super;">3 </span><span>in size.</span></div><div class = "text-block">A useful link: http://wiki.claritytechniques.org/index.php/Immunostaining </div></div>

[STEPS]
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Wash out residual clearing solutionThree washes in PBST, 24 hours each, 37C with gentle agitation.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Nonspecific Epitope BlockingBlocking buffer incubation: three days, 37C with gentle agitation.Note:  Serum type should be used based on the secondary antibody host species.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Primary Antibody Incubation1:100 dilution (~10 ug/mL) in Staining Buffer.Primary antibody incubation: five days, 37C with gentle agitation; then two days, 4C with gentle agitation.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Wash out Primary AntibodyThree washes, 24 hours each, at 37C with gentle agitation.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Secondary Antibody Incubation1:200 dilution (~5 ug/mL) in Staining Buffer. Secondary antibody incubation: five days at 37C with gentle agitation; two days at 4C with gentle agitation. Note: Protect samples from light.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Wash out Secondary AntibodyThree washes, 24 hours each, at 37C with gentle agitation. Note: Protect samples from light.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Prepare for 3D Imaging or StoragePrepare for imaging or store samples in washing buffer (PBST) at 4C. For 3D imaging guide, see: https://dx.doi.org/10.17504/protocols.io.begajbse