eamag/protocols-with-steps huggingface dataset:
TITLE: 3&#39; RNA Adapter Ligation to Input RNA
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.</div></div>

[STEPS]
?. [Anneal Adapter]
Take  (from Dephosphorylation of Input RNA).
[RNA]
?. [Anneal Adapter]
Add  and  (see Note 3).
[100% DMSO]
[InvRiL19 adapter]
?. [Anneal Adapter]
Incubate at  for . Place  >.
65 °C
on ice
?. [Prepare Ligation Master Mix; 13.5 μL Per Sample]
AB110× Ligase Buffer (with DTT)2.0 μL20.1 M ATP0.2 μL3Murine RNase Inhibitor0.2 μL4100% DMSO0.3 μL550% PEG 80008.0 μL6RNA Ligase high conc1.3 μL7H2O1.5 μL
AB110× Ligase Buffer (with DTT)2.0 μL20.1 M ATP0.2 μL3Murine RNase Inhibitor0.2 μL4100% DMSO0.3 μL550% PEG 80008.0 μL6RNA Ligase high conc1.3 μL7H2O1.5 μL
?. [Prepare Ligation Master Mix; 13.5 μL Per Sample]
Add  to each sample, mix, incubate at  for .  Flick to mix every ~.
[Ligation master mix]
0 Room temperature
?. [Silane Cleanup Input RNA]
Prepare beads:
?. [Silane Cleanup Input RNA]
Magnetically separate  per sample, remove the supernatant.
[MyONE Silane beads]
?. [Silane Cleanup Input RNA]
Wash 1× with  and resuspend beads in .
[ RLT buffer]
[ RLT buffer]
?. [Silane Cleanup Input RNA]
Bind RNA:
?. [Silane Cleanup Input RNA]
Add beads in  to sample, mix and add .
[RLT buffer]
[100% EtOH]
?. [Silane Cleanup Input RNA]
Pipette mix, leave pipette tip in a tube, pipette mix every ~  –5 min for .
?. [Silane Cleanup Input RNA]
Wash beads:
?. [Silane Cleanup Input RNA]
Magnetically separate, remove the supernatant.
?. [Silane Cleanup Input RNA]
Add , pipette resuspend and move to a new tube.
[75% EtOH]
?. [Silane Cleanup Input RNA]
After , magnetically separate, remove the supernatant, and wash 2× with 75% EtOH ().
?. [Silane Cleanup Input RNA]
Magnetically separate, remove residual liquid with fine tip → Air-dry .
?. [Silane Cleanup Input RNA]
Elute RNA:
?. [Silane Cleanup Input RNA]
Resuspend in , let it sit for .
[H2O]
?. [Silane Cleanup Input RNA]
Magnetically separate, transfer the supernatant to a new tube.
TITLE: NCEM Drop - Tissue Dounce Homogenisation (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
tissue dounce homogenisation

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR:



SECTION: Conventional
7. Adsorb 10 µL sample to grid 10 min , inspect to ensure sample does not dry out.

SECTION: Conventional
8. Drain excess sample from grid using filter paper, leave wet.
SECTION: Conventional
9. Stain  1 min 

10. Drain & dry using filter paper
SECTION: Dounce Homogenisation
2. This method is often used for scabs, warts, or chunks of tissue such as liver. Place ~ 5mm x 5mm piece of scab or tissue into glass homogeniser tube. 

SECTION: Dounce Homogenisation
4. Add equal volume TC water to tube, homogenise(20 strokes) inside Class II BSC cabinet.
SECTION: Dounce Homogenisation
3. Insert teflon plunger into cordless drill, set drill on max speed.
SECTION: Dounce Homogenisation
5. Clarify resultant solution at 13000 rpm  in Eppendorf centrifuge for 1 min 


SECTION: Dounce Homogenisation
6. Stand for 5 min to permit viruses to diffuse back into solution, from the debris, and provide an interface for the sampling of membrane associated viruses. Use supernatant for sample below.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A guide to reading and extracting key points from research articles. This protocol will suit the purposes of the assignments to summarize the readings for the class. Aim for roughly one page of typed text.</p>
<p> </p>
<p>Imagine presenting the contents of your summary (excluding the NOTES/BACKGROUND section) in 5 minutes. In other words, try to strike a middle ground between reading too little to be able to grasp the motivation/findings and reading too much and getting bogged down in the detailed minutiae. </p>

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

[DESCRIPTION]
<p>PreC-C Primers encompass described PreC-C mutations: mutation G1896A in the PreC region and mutations A1762T and G1764A in C-gene promotor. Primers were modified according to literature-based alignment, in order to optimize the amplification of all HBV genotypes. Sequence analysis at position 1762, 1764 et 1896 allows to determine sample wild type or mutant sequence (<em>i.e.</em> HBeAg expression or not, respectively). It is also possible to describe others rare punctual mutations. Furthermore, the sequence analysis also allows assessing HBV genotyping.</p>

[STEPS]
?.
TITLE: eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer
AUTHORS: Abigail Wells, Linda Park

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.</div></div>

[STEPS]
?. [DNA extraction]
heat-shock	Filters in  of at for and then allow samples to cool to room temperature
2 mL
95 °C
[min]
{"blocks":[{"key":"5iktf","text":"Lysis buffer recipe (Longmire et al 1997): ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"6ld12","text":"To make 1 liter","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [DNA extraction]
digestion	add  proteinase K (final concentration )
100 µl
?. [DNA extraction]
incubation	 at  for
56 °C
33
?. [DNA extraction]
phase lock set up	add ca.  of vacuum grease with a syringe onto the wall of the 5ml tube without touching the lysate to avoid cross-contamination
800 µl
?. [DNA extraction]
add 2 ml of phenol-chloroform-isoamyl (25:24:1) pH is critical for good results
?. [DNA extraction]
shake well and centrifuge
Centrifuge: 13300 34, 5 min, 4 10
?. [DNA extraction]
place ca.  of vacuum grease in 2 sets of empty tubes for CI. (These can be prefilled for convenience)
800 µl
?. [DNA extraction]
add  of chloroform:isoamyl 24:1 and decant aqueous layer from step 6.Important!! only add the chloroform just before use (while the tubes with PCI are in the centrifuge) so it does not affect the phase lock if grease is at the bottom.
2.2 mL
?. [DNA extraction]
shake well and centrifuge
Centrifuge: 13300 34, 5 min, 4 10
?. [DNA extraction]
?. [DNA extraction]
add of isopropanol (can be prefilled for convenience), add of  NaCl and decant aqueous layer from step 11
2 mL
80 µl
?. [DNA extraction]
invert several times
?. [DNA extraction]
precipitation	overnight (or 2 h) at room temperature
?. [DNA extraction]
centrifuge
Centrifuge: 13300 34, 30 min, 4 10
?. [DNA extraction]
pour liquid off slowly and add  of ice cold EtOH
800 µl
?. [DNA extraction]
shake well and centrifuge   and
Centrifuge: 13300 34, 5 min, 4 10
?. [DNA extraction]
pour liquid off slowly and allow tubes to dry for 1 h or until dry
?. [DNA extraction]
once they are dry, resuspend in  TE buffer (warm  to favor elution)
100 µl
37 °C
?. [DNA extraction]
store in the freezer ()
-20 °C
TITLE: IF Labeling Protocol
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the Labelling of Immunofluorescent Protein.

[STEPS]
SECTION: Immunofluorescent Protein Labeling Protocol
1. Alex Fluor 488 Microscale Protein Labelling Kit (Invitrogen, A30006).
SECTION: Immunofluorescent Protein Labeling Protocol
1.1. According to manufacturer protocol, AF 488 reactive dye has Tetrafluorophenyl (TFP) ester moiety that is more stable in solution than the commonly used succinimidyl (NHS) ester. TFP ester react efficiently with primary amines of protein to form a stable dye-protein conjugate and is independent of pH between 4 and 10. 

SECTION: Immunofluorescent Protein Labeling Protocol
1.2. Single kit can label a total of 200 µg of αS preformed fibrils.

SECTION: Immunofluorescent Protein Labeling Protocol
1.3. Dilute Hu-WT-αS fibrils (PFFs) to a concentration of 1 3189 in PBS followed by sonication (1 Sec ON/1 Sec OFF) for 2 min with 20% amplitude. 

SECTION: Immunofluorescent Protein Labeling Protocol
1.4. Mix diluted PFFs with component B (NaHCO3), then added freshly prepared reactive dye (component A) and incubated for 15 min at  Room temperature .

SECTION: Immunofluorescent Protein Labeling Protocol
1.5. Calculate amount of reactive dye using dye/protein molar ratio of 9.

SECTION: Immunofluorescent Protein Labeling Protocol
1.6. Remove excess dye by resin gel (component E) purification. 

SECTION: Immunofluorescent Protein Labeling Protocol
1.7. Place conjugated reaction mixture of PFFs and reactive dye on the center of the resin bed surface, centrifuge at 16000 x g, 15 s. 

SECTION: Immunofluorescent Protein Labeling Protocol
1.8. Collect the filtrate as labeled PFFs.
TITLE: Preparation of BSA complexed free fatty acids for in vitro studies
AUTHORS: Caroline  CT Tremblay, Julien  Ghislain, Vincent  Poitout

[DESCRIPTION]
This protocol describes the steps to prepare a fatty acid:BSA complex in cell culture media. It is suitable for the treatment of primary tissue (eg. isolated islets) and cell lines in ex vivo culture. Briefly, fatty acid stock solutions are prepared and complexed with BSA prior to dilution in culture media. Final fatty acid concentrations range from 0.125 to 0.5 mM. We recommend replacing the fatty acid mixture once each day throughout the treatment.

[STEPS]
SECTION: Preparation of BSA:FA complex
1. Preparation of Palmitate (PA) and Oleate (OA) stock solutions
As PA and OA are in powder form and very hydrostatic, handle with care as the  powder will stick to everything and move about readily.

Carefully weigh out a sufficient amount of each to make 150 millimolar (mM)  stock solutions in ethanol.
 
MW (PA) = 278.41, stored in a desiccator at 4 °C. 
MW (OA) = 305, stored in a desiccator at -20 °C.

Preparation of 1 mL of 150 millimolar (mM) stock solution: 

PA Weight out 41.8 mg and put in a 1.5 ml tube. 
Add 1 mL of 50 % (v/v) ethanol.  
Vortex and heat at65 °C until dissolved; vortex periodically.
 
OA Weight out 45.7 mg and put in a 1.5 ml tube. 
Add 500 µL of 100 % (v/v) ethanol. 
Vortex and heat at 65 °C for 15 min; mix occasionally. 
Then add 500 µL of MilliQ water, and vortex. The remaining  powder should dissolve instantly. 
Return to 65 °C until ready for use.
SECTION: Preparation of BSA:FA complex
2. Preparation of 0.5 mM fatty acid solution in culture media (FA:BSA at 5:1 molar ratio)

Prepare 10 % (v/v) fatty acid-free BSA: 
Weight 5 g and dissolve in50 mL of MilliQ water. 
Filter solution with a Millipore Steriflip-GP Sterile Centrifuge Tube Top Filter Unit.
Store at 4 °C.
Under a laminar flow hood, add the following components per ml of culture media: 
Place 67 µL of 10 % (v/v) fatty acid-free BSA into a 15 ml Falcon tube. 
Heat in 37 °C water bath for 5 min. 
Add 3.3 µL of the 150 millimolar (mM) fatty acid stock solution. 
Return to the 37 °C water bath for 5 min. Visually check for cloudiness. If clear, great, if cloudy, start over. 
Incubate in the 37 °C water bath for 60 min. 
Then add 930 µLof culture medium (eg. RPMI 1640 plus 10 % (v/v) FBS) heated to 37 °C. 
For control tube (Vehicle/BSA), add 3.3 µL of 50 % (v/v) ethanol to 67 µL of 10 % (v/v) fatty acid-free BSA into a 15 ml Falcon tube, and treat as above.
SECTION: Preparation of BSA:FA complex
3. Preparation of 0.15 mM fatty acid solution in culture media (FA:BSA at 1.5:1 molar ratio)
To prepare a solution with a final fatty acid concentration of 0.125 millimolar (mM) proceed as described in step 2 adding 1 µL of the 150 millimolar (mM) fatty acid stock solution (instead of 3.3 µL). Also reduce the ethanol to 1 µL for the control. 
To prepare any fatty acid concentration up to0.5 millimolar (mM) final adjust the amount of fatty acid stock solution accordingly while keeping the BSA constant.
TITLE: Environmental  Grab Sampling and Membrane Filtration
AUTHORS: Dilip  Abraham, Nicola Elviss, Nicholas Feasey, Nick  Grassly, Jacob John, Gagandeep Kang, John  Scott Meschke, Venkata  Raghava Mohan, Satheesh Nair, Jonathan  Rigby, Rajan Srinivasan, Catherine  Troman, Christopher  Uzzell, Nicolette Zhou

[DESCRIPTION]
A protocol for the collection of environmental samples (sewage and wastewater) using grab sampling and membrane filtration. This is part of a set of protocols for environmental surveillance of Salmonella Typhi. 

1L of sewage or wastewater is collected then filtered under a vacuum to capture the bacteria on filter discs ready for DNA extraction. 

This protocol is adapted from the method described in Rigby et al (2021).

[BEFORE_START]
Ensure that you wear appropriate PPE whilst sampling (e.g. gloves, appropriate footwear, disposable lab coat, facemask). 

[GUIDELINES]
It is recommended that sampling be done in the morning.

[STEPS]
SECTION: Sample Collection
1. Using a bucket or another method, fill a 1L collection container with sewage or wastewater and securely close the container.
SECTION: Sample Processing
5. Upon receipt in the lab the sample should be stored at 4-8ºC until filtration

SECTION: Sample Processing
6. Assemble the membrane filtration apparatus according to the manufacturer's instructions
SECTION: Sample Processing
7. Measure the sample into a large, sterile graduated cylinder
SECTION: Sample Collection
2. Wipe the outside of the collection container with disinfectant.
SECTION: Sample Processing
8. Place a 47mm 0.45µM filter disc on the filtration unit head and attach the filtration unit cup.

If necessary, place a coffee filter on top of the cup as a pre-filter to remove any larger solid matter.
SECTION: Sample Collection
3. Place the container in a cold container with ice packs for transport back to the laboratory
SECTION: Sample Collection
4. Place any disposable items in a biohazard bag for disinfection and disposal. Place any reusable items into a biohazard bag for return to the library and disinfection. Sanitise hands with hand sanitiser.
SECTION: Sample Processing
9. Pour sample into the filtration cup and turn on the vacuum to allow sample to slowly filter through the disc for 1 hour.

If the filter becomes clogged, pipette the remaining sample back into the measuring cylinder, and replace the filter disc and coffee filter with fresh filters. Repeat as necessary with up to 5 filters.
SECTION: Sample Processing
10. Using sterile forceps, transfer the filter discs to a small WhirlPak bag, add 10ml of Ringer's lactate and then seal the bag shut
SECTION: Sample Processing
11. Elute the filter discs by massaging the bag until the filters are clean or have fallen apart
SECTION: Sample Processing
12. Pipette 1ml of the eluate into a 1.5ml microcentrifuge tube and centrifuge at 10000 x g, 10 min 
Pipette off the supernatant and store the pellet at -20ºC until DNA extraction.

SECTION: Sample Processing
13. Thoroughly disinfect membrane filtration cups and measuring cylinders after use to avoid cross contamination of samples.
TITLE: Brain Slicing for Immunohistochemistry
AUTHORS: Angelica Martinez, McKenzie Pavlich, Caitlin Crook, Kokila Shankar, Giordano De Guglielmo, Olivier    George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol is used for sectioning brains in order to use in downstream IHC experiments.</div></div>

[STEPS]
?. Wash brain with 1x
?. Dispose of the current solution that brain is in into the proper waste stream
?. Transfer brain into with  of 1x PBS. Invert the tube to wash and pour out PBS.
20 mL
?. Repeat 2x for a total of 3 washes
?. Place cleaned brain flat onto petri dish. Using straight razor blade, cut of back half of cerebellum perpendicular to petri dish
?. Place the chuck onto dry ice. Place the brain in the center of the chuck with cerebellum on the bottom and use  to add 1 layer of glue on bottom edge of brain.
?. Once the first layer is frozen, repeat process 2x for a total of 3 layers of mounting media.
?. Insert chuck into holder with dorsal side of brain facing up. The front of the brain should be pointing directly at you. This is to create coronal sections.
?. Start turning the handle slowly and make adjustments to the positioning of the brain if needed. Make sure each slice is cutting evenly at the correct angle.
?. Slice until you get to the region of interest. Place glass cover onto blade to collect the slices
?. Collect ~5 slices and use a wet paint brush to carefully transfer the slices in the first vial. If you need to collect more than 5 slices for this region, continue collecting in groups of 5 and transfer each group into the next vial.
?. Repeat steps 5-6 to get slices from all the regions of interests.
?. When finished, store with vials with the slices in
4 °C
TITLE: Pythium Zoospore Production Soaking Solution
AUTHORS: Nimalka M Weerasuriya

[DESCRIPTION]
Creation of soaking solutions for Pythium myriotylum to be used for large-scale zoospore production.

[STEPS]
SECTION: Soaking Solutions
2. Make soaking solutions 1, 2 and 3:

 

SECTION: Preparation
1. Have mature colonies of verified Pythium myriotylum growing on CMA or 1.5-2% WA. Colony maturity ~7 days, with visible oospores.

 

SECTION: Soaking Solutions
2.1. Soaking Solution #1, 0.01 Molarity (m) (1 L):
1 L RO water at pH 7 in 1 L beaker
Add 1 g CaCO3
Filter through Whatman #1

pH is adjusted from pH 8.5  to pH 10.5 with  1 N  to duplicate soaking solution in original methods (Pacumbaba, cite)

SECTION: Soaking Solutions
2.2. Soaking Solution #2, 0.01 Molarity (m)+0.001 Molarity (m) (500 mL):
<missing info>

pH is adjusted to pH 7  with 1 N 
 

SECTION: Soaking Solutions
2.3. Soaking Solution #3, 0.001 Molarity (m) (500 mL):
<missing info>

pH is adjusted to pH 7  with 1 N 

SECTION: Soaking Solutions
3. Autoclave for 20 minutes liquid cycle
 

SECTION: Testing Zoospore Production
4. Take 30 mL of each Soaking Solution   onto surface of 7-day cultures in plate.

SECTION: Testing Zoospore Production
5. Incubate under light at Room temperature for 1440 min. 
Check for abundant sporangia that will appear after immersion.

SECTION: Testing Zoospore Production
6. At 1.5 up to 4 h every 30 minutes:
Take 80 µL from each immersion to a microcentrifuge tube with 20 µL. Gently invert to stain (or gently vortex) and immobilize zoospores.

SECTION: Testing Zoospore Production
7. Take 10 µL of liquid into haemocytometer and examine under 40x to quantify.
TITLE: P8 COMPROMISO DOCUMENTAL
AUTHORS: cgarcia

[DESCRIPTION]
La Universidad Católica establecerá las funciones de supervisión de los doctorandos mediante un compromiso documental firmado por: la universidad, el doctorando, su tutor y su director. Este compromiso será rubricado a la mayor brevedad posible después de la matriculación, y habrá de incluir un procedimiento de resolución de conflictos y contemplar los aspectos relativos a los derechos de propiedad intelectual o industrial que puedan generarse en el ámbito de los programas de doctorado.

[STEPS]
SECTION: P8 COMPROMISO DOCUMENTAL
1. El tutor, en la primera reunión mantenida con el doctorando tras su matriculación en el programa de doctorado, explicará al alumno como generar el documento descargando el modelo a través del Portal del Doctorando. En dicho documento se detallará la dedicación del doctorando al programa y el alumno deberá presentarlo a la firma de su tutor y director, una vez firmado deberá entregar el documento correctamente cumplimentado y firmado en la Secretaría de la Eiducam personalmente o escaneado a través del email tramitesdoctorado@ucam.edu
SECTION: P8 COMPROMISO DOCUMENTAL
2. En caso de establecer la dedicación parcial se deberá aportar la justificación correspondiente y La Comisión Académica de cada Programa deberá supervisar anualmente que se mantiene el motivo por el cual se concedió la parcialidad.
SECTION: P8 COMPROMISO DOCUMENTAL
3. La secretaría de la EIDUCAM se encargará de validar y custodiar el documento.
SECTION: P8 COMPROMISO DOCUMENTAL
4. Anualmente coincidiendo con la renovación de matrícula se consultará a los alumnos a efectos de actualizar la información respecto a la dedicación y a posibles cambios en la dirección o tutela del doctorando.
TITLE: Whole mount immunohistochemistry (WM-IHC)
AUTHORS: Marta   Wawrzyniak, Nathalie  Weber, Simon  Blanchoud

[DESCRIPTION]
This protocol has been successfully used with colonial tunicates has been adapted to these animals based on the following publication:

The onset of whole-body regeneration in Botryllus schlosseri: morphological and molecular characterization.  

[STEPS]
SECTION: Fixation
2. Anesthetize the animals following this protocol. See Whole colony fixation V.1.


SECTION: Fixation
3. Fix the animals in 4% PFA   at Room temperature . See Whole colony fixation V.1.



SECTION: Day 1
29. Wash in 3.3x PBS for 30 min and then in 1x PBS 60 min . Store at 4 °C .



SECTION: Day 1
29. 
SECTION: Fixation
1. Clean the slide with the colony of your interest. 
Cleaning colonial ascidians.
  
29. 
SECTION: Permeabilization and quenching
6. 
Place samples in PBST-SC.

SECTION: Permeabilization and quenching
7. Shake   at Room temperature . 



SECTION: Permeabilization and quenching
8. Rinse once with 1x PBS.
SECTION: Fixation
4. Wash twice in 3.3x PBS for 30 min .
SECTION: Fixation
5. Rinse quickly 3 times with 1x PBS and then leave in 1x PBS for 60 min .
SECTION: Blocking and staining
9. Place samples in PBST-GS.  
SECTION: Blocking and staining
10. Shake at Room temperature for 120 min .

SECTION: Blocking and staining
11. Stain your samples with primary antibody for 1800 min .

SECTION: Blocking and staining
11.1. Dilute the primary antibody in PBST-GS according to the manufacturer.
SECTION: Blocking and staining
11.2. On the slide, circle the system of interest with a PAP pen and wait 2 min for the circle to dry.

SECTION: Blocking and staining
11.3. Place the slide flat into the humidity chamber.
SECTION: Blocking and staining
11.4. Pipette the primary antibody solution on the circled system and incubate at 4 °C  for 1800 min .

SECTION: Blocking and staining
12. Wash four times in 1x PBST at Room temperature for 30 min slowly on a linear. 

SECTION: Blocking and staining
13. 
Stain your samples with secondary antiboby for 1800 min .
SECTION: Blocking and staining
13.1. Dilute the antibody in PBST-GS according to the manufacturer (keep solution and sample protected from the light!).
SECTION: Blocking and staining
13.2.  Incubate   in darkness at 4 °C in the humidity chamber. 

SECTION: Blocking and staining
14. Wash four times in 1x PBST at Room temperature for 30 min slowly on a linear shaker.  
SECTION: Blocking and staining
15. Dry as much as possible.
SECTION: Blocking and staining
16. Circle the sample with vaseline to build a custom-made chamber.
SECTION: Blocking and staining
18. Image your slides directly or store them at 4 °C .

SECTION: Blocking and staining
17. Mount with mounting medium, cover with a coverslip.