eamag/protocols-with-steps huggingface dataset:
TITLE: HPA Cell Atlas Standard Immunostaining Protocol
AUTHORS: Peter Thul, Ulrika Axelsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Cell Atlas, part of the Human Protein Atlas project, systematically investigate the spatiotemporal subcellular distribution of human proteins. This is our standard protocol for indirect immunostaining of cells cultured in 96-well glass bottom plates. </div></div>

[STEPS]
?. [Fixation]
Remove the growth medium (i.e. aspirate ~  from all wells).
80 µl
?. [Fixation]
Wash the cells once with 1X PBS.
40 µl
?. [Fixation]
Fix the cells by incubating in  4% PFA/1XPBS for 15 minutes.
40 µl
?. [Permeabilization]
Remove the PFA and permeabilize the cells by incubating with  0.1 % Triton X-100/1X PBS for 3 x 5 minutes.
40 µl
?. [Permeabilization]
Remove the Triton X-100 and wash the cells once with  1XPBS.
40 µl
?. [Primary Antibody Incubation]
Add blocking buffer with diluted primary antibodies; 2 ug/ml HPA antibody, 1 ug/ml anti-alpha tubulin (Abcam, Ab7291), and 1.25 ug/ml anti-calreticulin (Abcam, Ab2908). Incubate over night at 4°C or for two hours at room temperature. or
40 µl
[overnight]
[(RT)]
?. [Secondary Antibody Incubation]
Remove the blocking buffer with primary antibodies and wash the cells with 1X PBS for 4 x 10 minutes.
40 µl
?. [Secondary Antibody Incubation]
Add blocking buffer with diluted secondary antibodies; 2.5 ug/ml anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, A11034), 2.5 ug/ml anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific, A21424), and 2.5 ug/ml anti-chicken Alexa Fluor 647 (Thermo Fisher Scientific, A21449). Incubate at room temperature for 90 minutes while keeping dark.
40 µl
?. [DAPI Nuclear Staining and Mounting]
Remove the blocking buffer and incubate with 0.2 ug/ml DAPI/1X PBS  for 10 minutes.
40 µl
?. [DAPI Nuclear Staining and Mounting]
Wash the cells with 1X PBS for 4 x 10 minutes.
40 µl
?. [DAPI Nuclear Staining and Mounting]
Fill the wells with glycerol/10X PBS  and seal the plate with an adhesive aluminium PCR plate seal. Plates can be stored at 4°C for at least two weeks.
TITLE: Schistosoma mansoni cercariae transformation (without needle)
AUTHORS: Sarah K Buddenborg

[DESCRIPTION]
Free-living aquatic S. mansoni cercariae transform into the first intramammalian stage, called schistosomula or somules, by burrowing in the host skin. Upon contact, cercariae begin to enter the skin and lose their tails, becoming schistosomula. Somules migrate through the epidermis to the dermis to find a small venule or lymphatic vessel to enter the vasculature.  

Transformation of cercariae to schistosomula can be mimicked in the laboratory by centrifuging cercariae to remove tails and then culturing the somules in somule media. This method is particularly useful when the number of cercariae is low (i.e. clonal cercariae from an individual snail).

Somules can be cultured for several weeks with regular media changes.

[BEFORE_START]
Place 1 sterile 15ml falcon tube on ice per snail to be shed
Pre-chill benchtop centrifuge to 4°C
Prepare 1x DPBS+2% anti-anti and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Prepare somule media and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)

[GUIDELINES]
Media changes and opening of transformed somules to take place in tissue culture hood using sterile techniques

[STEPS]
SECTION: Cercariae collection
1. Shed cercariae from snails in a 6-well plate (see protocol "Schistosoma mansoni cercariae shedding"). The snails can be shed for up to 2 hrs by collecting cercariae and replacing with fresh water every 30 min
2. Using a sterile transfer pipette, dispense cercariae into sterile 15ml falcon tubes on ice

 

3. After collecting cercariae, adjust the volume in each 15ml falcon tube to 15ml with sterile water
4. Incubate tubes containing cercariae for 30 minon ice 

SECTION: Cercariae tail removal
5. Centrifuge the cercariae 2200 rpm, 30 min, 4 °C
6. Quickly remove the supernatant and resuspend the pellet in 10ml pre-warmed 1x PBS + 2% PSF (see "MATERIALS" section for recipes) by gently inverting the tube (do not use pipet to mix)
7. Centrifuge the cercariae  1500 rpm, 10 min, 4 °C 
8. Repeat steps 6 and 7 six more times

SECTION: Somules in culture
9. Resuspend pellet of somules in ~5ml pre-warmed 37 °C somule media (see "MATERIALS" section for recipes)

SECTION: Somules in culture
10. 
Place somules in a 6-well plate and top up each well with pre-warmed 37 °C somule media so each has a total of approximately 4ml media  

SECTION: Somules in culture
11. Incubate at 37 °C, 5% CO230 min

SECTION: Somules in culture
12. Optional: the following day, remove tails with transfer pipet (the tails will have floated to the top of the water column in the wells)
TITLE: Terrific broth (TB) medium
AUTHORS: Andreas Sagen

[DESCRIPTION]
IBI’s Terrific Broth is used with Glycerol in cultivating recombinant strains of E. coli. Terrific broth is a highly enriched medium for improving yield in plasmid bearing E. coli. Recombinant strains have an extended growth phase in the medium. The addition of tryptone and yeast extract in the medium will allow higher plasmid yield per volume. Glycerol is used as a carbohydrate source in this formulation. Unlike glucose, glycerol is not fermented to acetic acid.

[BEFORE_START]
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated

[GUIDELINES]
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.

[STEPS]
SECTION: Terrific broth base solution
1. Fill the bottle with 200 mL distilled water

 

SECTION: Terrific broth base solution
3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min
SECTION: Terrific broth base solution
5. Adjust pH while mixing to pH 7.2 using concentrated sodium hydroxide
SECTION: Terrific broth base solution
2. Measure 11.8 g Yeast extract and 5.9 g Tryptone. Add 7.5 g for making agar plates

Powdered compounds:
 
 
 
SECTION: Terrific broth base solution
7. Autoclave liquid at 121 °C for 15 min
SECTION: Terrific broth base solution
4. Add 100 mL Potassium phosphate buffer (1 M, 6.2 pH)
 

SECTION: Terrific broth carbon solution
8. Fill the bottle with 300 mL double-distilled water
SECTION: Terrific broth carbon solution
9. Add 20 mL Glycerol and mix solution well

Liquid compounds:
 
SECTION: Terrific broth carbon solution
10. Adjust pH while mixing to pH 7.2 using concentrated sodium hydroxide
SECTION: Terrific broth carbon solution
11. Add distilled water to a total of 500 mL
SECTION: Terrific broth carbon solution
12. Autoclave liquid at 121 °C for 15 minAutoclave liquid at 121 °C for 15 min
SECTION: 500 mL Terrific broth
13. Add 100 mL carbon solution to base solution
SECTION: Terrific broth base solution
6. Add distilled water to a total of 400 mL
SECTION: 500 mL Terrific broth
14. Aliquot medium in 100 mL units, and store refrigerated (4 °C)
TITLE: High Molecular Weight DNA Extraction Protocol [Tissue Sample]
AUTHORS: Aswini Leela

[DESCRIPTION]
This is an organic extraction protocol used for high molecular DNA extraction for tissue samples.

[STEPS]
SECTION: High Molecular Weight DNA Extraction Protocol
1. Lysis Tissue Sample
Add 500 µL of lysis buffer to 50 - 150 mg of   of sample. Make sure the tissue sample has been finely cut.

SECTION: High Molecular Weight DNA Extraction Protocol
2. Denatures and digest proteins that are subsequently hydrolyzed with Proteinase K
Add 15 µL of Proteinase K and vortex for 10 s . 

SECTION: High Molecular Weight DNA Extraction Protocol
3. Incubation Step
Incubate on a shaking incubator at 55 °C (150 min or till dissolved).

SECTION: High Molecular Weight DNA Extraction Protocol
4. Remove RNA with RNAse
Add 10 µL of RNAse and vortex briefly. 

SECTION: High Molecular Weight DNA Extraction Protocol
5. Incubation Step
Incubate in a shaking incubator for 10 min at 37 °C . 

SECTION: High Molecular Weight DNA Extraction Protocol
6. Partitioning of lipids and debris into an organic phase using P:C:IA
Add 525 µL of P:C:IA (25:24:1). Vortex until emulsion is formed.

SECTION: High Molecular Weight DNA Extraction Protocol
7. Centrifugation Step
Centrifuge at   

SECTION: High Molecular Weight DNA Extraction Protocol
8. Acquiring DNA in the Aqueous Phase
Pipette 400 µL of the aqueous layer into a new tube.

SECTION: High Molecular Weight DNA Extraction Protocol
9. Neutralize the charges on the sugar-phosphate backbone of the DNA with Sodium Acetate
Add 1/10th ( 20 µL ) 3M Sodium Acetate pH 5.2 .  Vortex gently (avoid creating bubbles). 

SECTION: High Molecular Weight DNA Extraction Protocol
10. Precipitation Step
Add 2 vol ( 440 µL ) of ice-cold 100% Ethanol. Mix gently and slowly by inverting the tube.

SECTION: High Molecular Weight DNA Extraction Protocol
11. Incubation Step
Incubate at -20 °C for 120 min .

SECTION: High Molecular Weight DNA Extraction Protocol
12. Centrifugation Step
Centrifuge at  . Discard supernatant carefully.

SECTION: High Molecular Weight DNA Extraction Protocol
13. Wash Step
Wash with 500 µL of ice cold 70% ethanol.

SECTION: High Molecular Weight DNA Extraction Protocol
14. Centrifugation Step
Centrifuge   

SECTION: High Molecular Weight DNA Extraction Protocol
15. Repeat Wash Step (Step 17 and 18) 2 more times. 

SECTION: High Molecular Weight DNA Extraction Protocol
16. Quick Spin the tube one more time and pipette any remaining ethanol out of the tube.
SECTION: High Molecular Weight DNA Extraction Protocol
17. Dry
Air dry for at least 120 min 

SECTION: High Molecular Weight DNA Extraction Protocol
18. Final Elution
Add 35 µL of elution buffer (EB) and gently mix by tapping.

SECTION: High Molecular Weight DNA Extraction Protocol
19. Storage
Store at 4 °C .
TITLE: Bedding Test
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Used to assess nigrostriatal sensorimotor function in mice. Based on the Deacon 2006 protocol.

[STEPS]
2. Provide few pellet of food (Standard diet, SDS, R1/CRM/R3) (around 2-3 pellets for a total of 5g) as well as water ad libitum.
1. Transfer the mice as single cage into a “Green line” cage (Techniplast, USA).
3. Place a 5 x 5 cm cellulose paper tissue folded in the right corner of the cage.
4. Place the cage in a ventilated rack with temperature, humidity and light control for 48h.
5. At the end of the 48h check the animal cage and take a picture.
6. Score the bedding as follow:
 0.    The mice did not touch the paper square.
   The mice unfolded the paper square but did not tear it up.
   The mice unfolded and started to tore up <10% of the paper square.
    The mice tore up the paper square <50%
    The mice tore up the paper square, but the bedding is not perfect.
    Perfect bedding
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Data Preparation
?. 
SECTION: Data Preparation
?. 
SECTION: Data Preparation
?. 
SECTION: Data Preparation
?. 
SECTION: Data Preparation
?. 
SECTION: Data Preparation
?. 
SECTION: Non-rigid Registration
?. 
SECTION: Non-rigid Registration
?. 
SECTION: Non-rigid Registration
?. 
SECTION: Non-rigid Registration
?.
TITLE: Making Quenching and Blocking Solution
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to make Quenching solution and Blocking solution used for protein EDAC particle coupling. Glycine serves as a primary amine source for quenching the coupling reaction. Bovine serum albumin (BSA) is used to coat particle surface to prevent nonspecific (e.g. hydrophobic) interaction. Adapted from Bangs Laboratories Inc. TechNote 205 Covalent Coupling.</div></div>

[STEPS]
?. [Make Quenching Solution]
Add  BSA to  DW to make solution.Swirl to dissolve BSA.
100 mg
100 mL
Recommended concentration from Tech Note 205 is   -  BSA. Volume and concentration can be adjusted as necessary.
?. [Make Quenching Solution]
Add  glycine to make  solution.
263 mg
Recommended concentration from Tech Note 205 is   -   glycine. Volume and concentration can be adjusted as necessary.
?. [Make Blocking Solution]
Add  BSA to  DW to make solution.Swirl to dissolve BSA.
1 g
100 mL
?. [Store Solutions]
Store solutions at  .
4 °C
TITLE: Quick protocol for protein extraction from adherent fish-derived fibroblasts
AUTHORS: Joao M Moreno, Vitor C Sousa, Romana Santos

[DESCRIPTION]
We present a rapid and efficient protocol for extracting total proteins from adherent fish-derived fibroblasts, specifically optimized for applications in Western blotting and mass spectrometry. The method involves directly dissociating cells from the culture flask into a lysis buffer (e.g. RIPA buffer) followed by centrifugation to collect the total soluble proteins. The use of buffers like RIPA ensures comprehensive solubilization of cellular proteins while preserving their integrity and functionality. This straightforward and reproducible protocol yields high-quality protein extracts suitable for various downstream analytical techniques. Its simplicity and reliability make it an invaluable tool for researchers studying proteomics using fish cell culture.

[STEPS]
1. Carefully remove all culture media from the flask and add enough ice-cold 1X PBS to wash the cells
2. Carefully remove the ice-cold 1x PBS and add ice-cold lysis buffer (RIPA buffer) according to the estimated number of cells:
1 mLfor    cells (roughly a T75 flask).

3. Use a cell scraper to dissociate the cells from the bottom of the flask.
4. Resuspend the cells in the lysis buffer and transfer the suspension to a microcentrifuge tube. 
6. Centrifuge at 13,000 x g for 20 min at 4°C.
7. Carefully transfer the supernatant containing the soluble protein to a new tube and keep on the ice. Discard the pellet.

Note: The protein solution can be kept in the freezer for longer storage periods until further use.
5. Agitate for 20 minutes at 4ºC.
TITLE: High Efficiency Transformation Protocol (C2987I)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the protocol for C2987I cells. If you are using the C2987H cells, please refer to this protocol.

[GUIDELINES]
Transformation Protocol Variables

Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.

Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.

Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.

Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

DNA Contaminants to Avoid
   Contaminant Removal Method   Detergents Ethanol precipitate   Phenol Extract with chloroform and ethanol precipitate   Ethanol or Isopropanol Dry pellet before resuspending   PEG* Column purify or phenol/chloroform extract and ethanol precipitate   DNA binding proteins (e.g. Ligase) Column purify or phenol/chloroform extract and ethanol precipitate   



[STEPS]
1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear.  
 

2. Mix gently and carefully pipette 50 µL into a transformation tube on ice. 
3. Add 1 µL-5 µL containing 1 pg-100 ng of plasmid DNA to the cell mixture.
4. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
5. Place the mixture on ice for 30 min. Do not mix. 
 

6. Heat shock at exactly 42 °C for exactly 30 s. Do not mix. 
 

7. Place on ice for 5 min. Do not mix.
8. Pipette 950 µL into the mixture.
9. Place at 37 °C for 60 min, shaking vigorously (250 rpm) or rotating. 
 

10. Warm selection plates to 37 °C.
 

11. Mix the cells thoroughly by flicking the tube and inverting.
12. Perform several 10-fold serial dilutions in SOC.
13. Spread 50 µL-100 µL of each dilution onto a selection plate.
14. Incubate 60 min at 37 °C.
TITLE: NCEM Drop Agar (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
drop agar

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR:



SECTION: Adsorption &amp; Staining
4. Place 5 µL sample on surface of agar square. Immediately float carbon coated formvar filmed grid face down on the droplet and leave until the droplet diffuses into the agar. 

SECTION: Adsorption &amp; Staining
5. Stain  1 min 

SECTION: Adsorption &amp; Staining
6. Drain & dry using filter paper
SECTION: Agar preparation
2. Make 2 Mass / % volume   in TC Water. Microwave to dissolve, pour into3 mL of the solution into 5 mL petri dishes and place in fridge to solidify. 

SECTION: Agar preparation
3. Cut 1 x1 cm squares out of agar with scalpel,  lift out onto filter paper
TITLE: Immunoblot analyses for investigating SpLA binding to purified mammalian and avian immunoglobulins.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. Aliquots of 2 µg/µl purified immunoglobulins from birds, laboratory, wild, farm animals and pets are applied to the gels of SDS-PAGE as described elsewhere.
?. Gels are transferred to nitrocellulose membranes (Immobilon-Nc, pore size 0.45 µm, Sigma-Aldrich Co, St Louis, Missouri) during 75 minutes at 40 mAmps using a semi-dry electroblotter, HEP-1 Model, Owl Scientific Inc.
?. The running buffer contains 25 mM Tris, 192 mM glycine pH 8.3 and 20% methanol.
?. The nitrocellulose membranes are blocked overnight in 10% non-fat skim milk in PBS with 0.05% Tween-20 pH 7.4 and then washed 4x, 10 minutes with PBS-Tween 20.
?. Recombinant protein LA (Sigma-Aldrich Co, St Louis, Missouri) at a concentration of 5 µg/ml is added to the membranes.
?. After that there is an incubation period at 4°C overnight.
?. The nitrocellulose membranes were washed as above.
?. A secondary antibody (rabbit anti-chicken IgY horseradish peroxidase, Sigma Aldrich) is added at a 1:15 000 dilution.
?. It is incubated for one hour at room temperature and washed as above.
?. Tetramethyl-benzidine (TMB) solution is added to the nitrocellulose membranes, which are then incubated in the dark for a few minutes.  Then, the membranes are shaken gently and rinsed thoroughly in de-ionized water to stop the blotting process and are left to dry.
?. Alternatively, Ig samples are transferred to nitrocellulose membranes and directly probed using SpLA-HRP (diluted 1:5000) and TMB (this system was mainly used for detecting avian Igs).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Rapid, cheap, and effective DNA extraction for PCR. Easily scaled up to 96-well plates. This originates with Truett et al (2000) <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=10907076%5Buid%5D" target="_blank">PMID:10907076</a></p>
<p> </p>
<p>This protocol is very well tested by many labs for subsequent PCR amplification. For PCR it does not matter that DNA also has protein, is in low concentration, and is fragmented. This is a very robust DNA preparation method.</p>
<p>It is not difficult to do 960 DNA extractions per day (10 plates), the major determinant is tissue handling.</p>
<div title="Page 1">
<div>
<p> </p>
</div>
<table style="width: 483px;">
<tbody>
<tr>
<td style="width: 248px;"> <strong>Alkaline Lysis Solution (pH 12)</strong></td>
<td style="width: 235px;">
<div>
<div>
<p style="text-align: left;">For 100 ml add</p>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 248px;">
<div>
<div>
<p>25 mM NaOH</p>
</div>
</div>
</td>
<td style="width: 235px;">
<div>
<div>
<p>25 ml of 100 mM NaOH</p>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 248px;">
<div>
<div>
<p>0.2 mM Na2EDTA</p>
</div>
</div>
</td>
<td style="width: 235px;">
<div>
<div>
<p>0.4 ml of 50 mM Na2EDTA (pH 8)</p>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 248px;"> </td>
<td style="width: 235px;">
<div>
<div>
<p>74.6 ml of ddH2O</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<table style="width: 481px;">
<tbody>
<tr>
<td style="width: 243px;"><strong> Neutralizing Solution (pH 5)</strong></td>
<td style="width: 238px;">
<div>
<div>
<p style="text-align: left;">For 100 ml add:</p>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 243px;">
<div>
<div>
<p>40 mM Tris-HCl</p>
</div>
</div>
</td>
<td style="width: 238px;">
<div>
<div>
<p>630 mg of Tris-HCl</p>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 243px;"> </td>
<td style="width: 238px;">
<div>
<div>
<p>100 ml of ddH2O</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p>Notes:</p>
<ul>
<li>Prepare alkaline lysis solution fresh each day</li>
<li>Neutralizing solution is stable at room temperature for long periods (months-years)</li>
<li>Tris-HCL is NOT Tris-base</li>
<li>The main cause of failure is too much tissue for the volume of alkaline lysis solution</li>
</ul>
</div>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Pig- Heart Neuonal and Fiber Immunocytochemistry
AUTHORS: Pradeep Rajendran, John Tompkins, Kalyanam Shivkumar

[STEPS]
?. Animal: Yucatan Minipig 1427-Gender: Male-Weight: 28.4Kg
?. Tissue Fixation:-Flush heart with PBS, RT,  and heparin then inject 4% paraformaldehyde in PBS into coronaries. -Removed heart and fix overnight at 4 degrees C in 4% PFA/PBS.-Transfer to container with 0.01M PBS+0.02M Na Azide+20% Sucrose on 6/15/2018..
?. Tissue Processing:-Cut the right atrial ganglionated plexus (RAGP) out of heart.-Rinsed tissue with 1XPBS, then froze in (-80) freezer.- Used O.C.T medium to mount RAGP on specimen plate in cryostat. -Cut RAGP at 30um at (-25) degrees Celsius. -9 slides x 8 sets-2 tissue sections per slide-Make sure the cut sections have completely adhered to the slides (approx 30 min) before wrapping the slide boxes in foil and placing in (-20) freezer.
?. Fluorescence Immunohistochemistry:Primary Antibodies:1.) VAChT-Synaptic Systems (SYSY): Cat.No.139103-Dilution: (1:500)-Host: rabbit2.) Anti-Tyrosine Hydroxylase-Millipore: Cat. #AB1542-Dilution: (1:500)-Host: sheep
?. Secondary Antibodies:1.) Alexa Fluor 488-conjugated AffiniPure Donkey Anti-Rabbit IgG-Jackson ImmunoResearch Lab: Code No. 711-545-152-Dilution: (1:200) 2.) Biotin-SP-conjugated AffiniPure Donkey Anti-Goat IgG -Jackson ImmunoResearch Lab: Code No.705-065-147 -Dilution: (1:200) 2.pt 2) Cy 3-conjugated Streptavidin - Jackson ImmunoResearch Lab: Code No. 016-160-084 -Dilution: (1:900)