eamag/protocols-with-steps huggingface dataset:
TITLE: UC Davis - Adiposity
AUTHORS: Jon Ramsey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">The major fat pads of mice will be dissected and weighed. This approach provides a way to determine if an intervention has an impact on specific depots of fat.</div></div>

[STEPS]
?. Body weight of animal is taken before anesthesia.
?. The mouse is euthanized according to the methods approved in the study’s IACUC protocol.
?. A midline incision is made to open the abdominal cavity.
?. Forceps are used to pull up the epididymal fat pad (males). The fat pad is cut just above the epididymis and removed.
?. The parametrial fat pad (females) is cut at the base of the uterus and separated from the horn and ovary.
?. The kidneys are pulled toward the midline to allow the retroperitoneal fat pad to be visualized. The method of Johnson and Hirsch is followed, except fat that extends past the lower pole of the kidney is also removed if it is not surrounding the kidney (perirenal).
?. The kidneys are removed from the abdominal cavity and the white fat surrounding each kidney is dissected.
?. Using fingers, the mesenteric fat is stripped from the duodenum to the colon.
?. After all abdominal fat pads are dissected, the animal is skinned and any fat between the muscle and skin is carefully dissected.
?. Adipose tissue in the intrascapular region is dissected. Care is taken to remove white adipose tissue covering the brown adipose tissue. The brown adipose depot is dissected and cleaned of visible white adipose tissue.
?. Each tissue’s weight is recorded. Left and right pad weights are combined for total pad weight.
?. Total white adipose tissue (WAT) is calculated by summing the weights of the gonadal (epididymal or parametrial), retroperitoneal (including perirenal), mesenteric, and subcutaneous depots. % body fat can be calculated by dividing wt of fat tissue by body weight of animal.
TITLE: Amino Acid Table
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Amino acid and DNA Bases table</div></div>

[STEPS]
?. [Amino Acids and DNA Bases]
Alanine0.5% (5 mg/ml)waterART/4°C40800Arginine HCI1% (10 mg/ml)waterART/4°C20200Asparagine0.5% (5 mg/ml)waterART/4°C40800Aspartic Acid1% (10 mg/ml)0.2 N NaOHFRT1001000Cysteine0.5% (5 mg/ml)waterFRT/4°C40800Glutamine2% (20 mg/ml)waterART/4°C40200Glutamic Acid1% (10 mg/ml)0.2 N NaOHFRT1001000Glycine2% (20 mg/ml)waterA4°C40200Histidine HCI1% (10 mg/ml)waterART/4°C20200Isoleucine1% (10 mg/ml)waterA4°C30300Leucine1% (10 mg/ml)waterART30300Lysine HCI1% (10 mg/ml)waterART/4°C30300Methionine1% (10 mg/ml)waterART/4°C20200Phenylalanine1% (10 mg/ml)waterART50500Proline2% (20 mg/ml)waterART/4°C40200Serine8% (80 mg/ml)waterART/4°C400500Threonine4% (40 mg/ml)waterART200500Tryptophan1% (10 mg/ml)0.1 N HCIF4°C (It. sens)20200Tyrosine0.2% (2 mg/ml)0.2 N NaOHFRT(It. sens)301500Valine3% (3 mg/ml)waterART/4°C150500Adenine Sulfate0.2% (2 mg/ml)0.1 N HCIFRT20StpCytosine0.5% (5 mg/ml)waterA4°C40800Guanine0.5% (5 mg/ml)0.1 N HCIF4°C40800Thymus0.1% (1 mg/ml)waterA4°C404000Uracil0.2% (2 mg/ml)waterART201000Uridine1% (10 mg/ml)waterFRT30300
Alanine0.5% (5 mg/ml)waterART/4°C40800Arginine HCI1% (10 mg/ml)waterART/4°C20200Asparagine0.5% (5 mg/ml)waterART/4°C40800Aspartic Acid1% (10 mg/ml)0.2 N NaOHFRT1001000Cysteine0.5% (5 mg/ml)waterFRT/4°C40800Glutamine2% (20 mg/ml)waterART/4°C40200Glutamic Acid1% (10 mg/ml)0.2 N NaOHFRT1001000Glycine2% (20 mg/ml)waterA4°C40200Histidine HCI1% (10 mg/ml)waterART/4°C20200Isoleucine1% (10 mg/ml)waterA4°C30300Leucine1% (10 mg/ml)waterART30300Lysine HCI1% (10 mg/ml)waterART/4°C30300Methionine1% (10 mg/ml)waterART/4°C20200Phenylalanine1% (10 mg/ml)waterART50500Proline2% (20 mg/ml)waterART/4°C40200Serine8% (80 mg/ml)waterART/4°C400500Threonine4% (40 mg/ml)waterART200500Tryptophan1% (10 mg/ml)0.1 N HCIF4°C (It. sens)20200Tyrosine0.2% (2 mg/ml)0.2 N NaOHFRT(It. sens)301500Valine3% (3 mg/ml)waterART/4°C150500
Adenine Sulfate0.2% (2 mg/ml)0.1 N HCIFRT20StpCytosine0.5% (5 mg/ml)waterA4°C40800Guanine0.5% (5 mg/ml)0.1 N HCIF4°C40800Thymus0.1% (1 mg/ml)waterA4°C404000Uracil0.2% (2 mg/ml)waterART201000Uridine1% (10 mg/ml)waterFRT30300
TITLE: Rotarod
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the Rotarod behavioural test.

[STEPS]
1. Habituate mice in the testing room for 60 minutes.
2. Place each mouse on the rod and turn on the rotarod machine at 4-40rpm over 5 minutes. Record how long each mouse can stay on the pole.
3. Once testing is complete, place mice back into their home cage and wait for 10 minutes.
4. Repeat this for 4 trials per day then repeat for 3 days (12 trials total).
TITLE: Synthetic media (A1) for Aurantiochytrium limacinum (ATCC MYA-1381)
AUTHORS: Anbarasu Karthikaichamy, Jackie Collier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Synthetic media recipe for </span><span style = "font-style:italic;">Aurantiochytrium limacinum </span><span>(ATCC MYA-1381). This media may not be suitable for </span><span style = "font-style:italic;">Aplanochytrium </span><span>and</span><span style = "font-style:italic;"> Oblongichytrium. </span></div></div>

[STEPS]
?. [5x A1stock]
Measure and add the following components to an appropriate media bottle. Make up the solution to desired volume with water. Autoclave for  at 15 PSI  and   AB1Componentsg/L2CaCl2·2H2O2.53Glycerol1504KCl&nbsp;25KH2PO4&nbsp;206maleic acid&nbsp;297MgCl2&nbsp;48Na2SO4&nbsp;159NaCl7210Sodium-glutamate hydrate12511Tris base30
121 °C
AB1Componentsg/L2CaCl2·2H2O2.53Glycerol1504KCl&nbsp;25KH2PO4&nbsp;206maleic acid&nbsp;297MgCl2&nbsp;48Na2SO4&nbsp;159NaCl7210Sodium-glutamate hydrate12511Tris base30
The composition is for 5X
?. [Trace metal mix]
Measure and add the following components to an amber bottle. Make up the solution  to desired volume with water. Filter sterilize and refrigerate the trace metal mix.   AB1Componentsg/L2FeSO4 · 7H2O503CuSO4 · 5H2O3.94ZnSO4 · 7H2O4.45MnSO4 · H2O1.56Na2MoO4 · 2H2O0.17CoCl2 · 6H2O in 0.6 M HCl0.2
AB1Componentsg/L2FeSO4 · 7H2O503CuSO4 · 5H2O3.94ZnSO4 · 7H2O4.45MnSO4 · H2O1.56Na2MoO4 · 2H2O0.17CoCl2 · 6H2O in 0.6 M HCl0.2
Do not autoclave!
?. [Vitamins]
Measure and add the following components to individual amber bottles. Make up the solution  to desired volume with water. Filter sterilize the vitamins separately and refrigerate.  AB1Componentsg/L2Thiamine·HCl (B1)5.00E-053Cyanocobalamin (B12)5.00E-06
AB1Componentsg/L2Thiamine·HCl (B1)5.00E-053Cyanocobalamin (B12)5.00E-06
Do not autoclave!
?. [Prepare working stock (1X)]
For ,  Add the stock solutions to an autoclaved bottle.  AB1Stock solutionVolume (mL)2A1 stock (5X )2003Vitamin B1 (1000X)14Vitamin B12 (10000X)0.15Trace metal mix (400X)2.5Make up the media to  using autoclaved Millipore water.
1 L
AB1Stock solutionVolume (mL)2A1 stock (5X )2003Vitamin B1 (1000X)14Vitamin B12 (10000X)0.15Trace metal mix (400X)2.5
1 L
To prevent bacterial contamination, add   of 100 mg/mL Ampicillin to the final media.One of the vitamins may not be added for the A1 vitamin drop out media.
?. [A1 plates]
Prepare 1X stock of A1 media (do not add trace metal and vitamins).Add 1% (w/v) agar in to the 1X A1 media.Autoclave for   at 15 PSI  and  . Allow the solution cool down.Add the trace metal mix, vitamins and appropriate antibiotics.Pour the plates and store it in the refrigerator until use. Add 1% (w/v) agar in to the 1X A1 media.Autoclave for   at 15 PSI  and  . Allow the solution cool down.Add the trace metal mix, vitamins and appropriate antibiotics.Pour the plates and store it in the refrigerator until use. Autoclave for  at 15 PSI  and . Allow the solution cool down.Add the trace metal mix, vitamins and appropriate antibiotics.Pour the plates and store it in the refrigerator until use.
121 °C
TITLE: Chiral Analysis
AUTHORS: BOC       Sciences

[DESCRIPTION]
Chirality plays an important role in the fields of asymmetric synthesis, biology and medicine. Therefore, it is of great significance to analyze chiral compounds quickly, sensitively, and selectively. BOC Sciences can perform chiral analysis following regulatory standards. We offer chiral column screening, chiral analysis method development and optimization services. Our chiral analysis experts can characterize the enantiomers of chiral compounds for your research needs.


[STEPS]
TITLE: Motility Medium(Buffer)
AUTHORS: Erçağ Pinçe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">MM for bacterial motility experiments</div></div>

[STEPS]
?. [Prepare Stock Solutions]
Potassium Phosphate(KPO4) Stock Buffer [1M] (100x):1-) Prepare 1M Potassium Phosphate Monobasic - 500 mL ddH2O (milliQ)- 68.045 gr of Monobasic potassium phosphate (KH2PO4) [1M]- 87.09 gr of Dibasic potassium phosphate (K2HPO4) [1M] 2-) Adjust the pH of the solution to 7.0 3-) Filter sterilize.4-) Store at room temperature(+4C).L-methionine Stock Buffer [10mM] (10000x):1-) Mix the ingredients- 500 mL ddH2O (milliQ)- 746 mg L-methionine 2-) Stir the mix until L-methione dissolve. 3-) Check the pH and adjust to 7.04-) Filter sterilize.5-) Store in the cold room(+4C).Note: You have to dilute this stock solution down to 100µM before adding into motility medium mix. Lactic Acid Stock Buffer [1M](100x):1-) Mix the ingredients- 46.27 mL ddH2O (milliQ)- 3.73 mL Lactic Acid (d =  1.209 g/mL) 2-) Briefly stir 3-) Check the pH and adjust to 7.04-) Filter sterilize.5-) Store in the cold room(+4C).  EDTA Stock Buffer[10mM](100x):1-) Mix the ingredients - 49 mL ddH2O (milliQ)- 1 mL EDTA [0.5M]2-) Briefly stir. 3-) Filter sterilize. 4-) Store in the cold room(+4C).
Since the masses of monobasic and dibasic potassium phosphate are large, they take up considerable amount of volume inside the buffer. To get the correct molar concentration of both compounds inside the buffer, first add chemicals inside an empty beaker and only then add the ddWater and complete the total volume of the buffer to 500mL.
?. [Prepare Motility Medium]
1-) Mix the following ingredients: - 480 mL ddH2O(milliQ)- 5 mL Potassium Phosphate(KPO4) [1M] (100x)- 5 mL L-Methionine [100 µM] (100x)- 5 mL Lactic Acid [1M] (100x)- 5 mL EDTA [10mM] (100x) - 67 µL NaCl [1M] 2-) Briefly stir. 3-) Adjust the pH to 7.0 4-) Filter sterilize. 5-) Store in the cold room(+4C).
The stock solution of L-Methionine is 1mM(1000µM). You must dilute this down to 100µM before adding into the motility buffer.
TITLE: Nuclei Isolation and Sorting from Frozen Human Temporal Cortex
AUTHORS: Clemens Scherzer

[DESCRIPTION]
This protocol is about nuclei isolation and sorting from frozen human temporal cortex.

[BEFORE_START]
Prepare Buffers as described in section 'Materials'.

[STEPS]
SECTION: Method
1. Cut 100 mg - 200 mg  into small pieces on dry ice.
SECTION: Method
2. Put cut tissue into a microcentrifuge tube (pre-cooled on dry ice), weigh in a balance.
SECTION: Method
3. Transfer tissue to a glass dounce tissue grinder on regular ice.
SECTION: Method
4. Add 2 mL  to the tissue grinder.
SECTION: Method
5. Homogenize tissue with pastel A 10 times and pastel B 10 times.
SECTION: Method
6. Transfer the homogenate into a 2 ml  microcentrifuge tube.
SECTION: Method
7. Incubate on ice  for 5 min, mix with Wide bore tips once during the incubation.
SECTION: Method
8. Filter the homogenate with a 70 μmSstrainer.
SECTION: Method
9. Centrifuge at 500 x g, 5 min, 4 °C.
SECTION: Method
10. Remove the supernatant and resuspend the pellet in 1.5 mL with Wide bore tips.
SECTION: Method
11. Incubate on ice for 5 min .
SECTION: Method
12. Centrifuge at 500 x g, 5 min, 4 °C.
SECTION: Method
13. Remove the supernatant and add 500 µL to the pellet without resuspension.
SECTION: Method
14. Incubate on ice  for 5 min.
SECTION: Method
15. Add 1.0 mL to the tube and resuspend the  pellet.
SECTION: Method
16. Centrifuge at 500 x g, 5 min, 4 °C.
SECTION: Method
17. Remove the supernatant and resuspend the pellet in 1.8 mL.
SECTION: Method
18. Pipette 200 µL from original tube (T1) into a new tube (T2).
SECTION: Method
19. Centrifuge both T1 and T2 at 500 x g, 5 min, 4 °C.
SECTION: Method
20. Remove supernatant in T1 and resuspend pellet with 500 µL.
SECTION: Method
21. Remove supernatant in T2 and resuspend pellet with 65 µL.
SECTION: Method
22. Filter nuclei in T1 and T2 with 35 μm strainer respectively.
SECTION: Method
23. Check nuclei under microscope and count the number with cell counter (Optional).
SECTION: Method
24. Add 2 mL to T1, mix by pipetting.

SECTION: Method
25. Add 240 µL to T2, mix by pipetting.
SECTION: Method
26. Use T2 as a negative control for sorting.
SECTION: Method
27. Sort nuclei from P3 gate into a 1.5 ml microfuge tube preloading 15 µL.

SECTION: Method
28. Collect 90,000 events from P3 gate, the final volume is about 40 µL - 60 µL.
SECTION: Method
29. Centrifuge at 500 x g, 5 min, 4 °C.
SECTION: Method
30. Remove supernatant and resuspend pellet with 15 µL.
SECTION: Method
31. Use 8.4 µL (50,400 events) for 10x GEM generation and barcoding following manufacture´s protocol.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
How to run Megahit version 1.1.3 (Li et al. 2015) through the iMicrobe plaform.
 
Megahit is a de-novo assembler for large and complex metagenomics NGS reads relying on succinct de Bruijn graph (SdBG) to achieve low memory assembly.
 
More informations about Megahit can be found here : https://github.com/voutcn/megahit

[BEFORE_START]
You need a working Cyverse account to connect to iMicrobe.To explore how to log into iMicrobe, read the dedicated protocol. 


Your dataset of interest should be metagenomic reads, in a fasta or fastq format.


In iMicrobe, there is several ways to run an app on a dataset (from the cart, from your personnal datastore and form an URL). If you need more information on how to run an app, read the protocol associated. This app is currently not available to run from the cart.

[GUIDELINES]
More informations and details about Megahit can be found there : https://github.com/voutcn/megahit
 
 
Several parameters are available to the user through the iMicrobe app. 
Choosing k
MEGAHIT uses multiple k-mer strategy. Minimum k  (--k-min) , maximum k (--k-max) and the step for iteration (--k-step) can be set by options. Please not that k must be odd numbers while the step must be an even number.
Filtering (Kmin+1)-mer
The parameter --min-count allows the user to discard (kmin+1)-mer with multiplicity lower than d . By default that parameter is set to 2. For high sequencing depth coverage (>40x), this value should be set to 3.
mercy k-mer
This tool is specially designed for metagenomics assembly to recover low  coverage sequence. For generic dataset >= 30x,  MEGAHIT may generate  better results with using the --no-mercy option.
K-min-1 pass mode 
This mode is more memory efficient for ultra low-depth datasets. It can be activated by using the option --kmin-1pass


[STEPS]
SECTION: De Novo assembly of paired-end illumina dataset
?. 
SECTION: De Novo assembly of 454 datasets
?.
TITLE: Combinatorial selective ER-phagy remodels the ER during neurogenesis
AUTHORS: Melissa Hoyer, Cristina Capitanio, Ian R. Smith, Julia C. Paoli, Anna Bieber, Yizhi Jiang, Joao A. Paulo, Florian Wilfling, Brenda A. Schulman, Harper JW

[DESCRIPTION]
The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis: a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodeling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of ER and the role of individual ER-phagy receptors is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodeling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy.  Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.




[STEPS]
TITLE: Ultracentrifugal separation of HDL alone and calculation of non-HDL
AUTHORS: Daniel Teupser, Jan Breslow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol is used to isolate the various lipid fractions from blood plasma using ultracentrifugation. The actual measured concentrations are performed separately once the isolations are complete.</div><div class = "text-block"><span style = "font-weight:bold;">NOTE:</span><span style = "font-weight:bold;font-style:italic;"> </span><span style = "font-style:italic;">This protocol IS applicable for ApoE knockout mice.</span></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqq6.jpg" /></div></div>

[STEPS]
?. Add 60 ul of plasma to Beckman ultracentrifugation tube (7 x 20 mm; thick walled; polyallomer; cat. # 343621).
?. Layer 60 ul of PBS on top of the plasma and place tubes in a TLA100 rotor.
?. Spin for 3 hours Beckman Optima TL tabletop ultracentrifuge at 70,000 rpm, 4°C.
?. Using a 100 μl Hamilton syringe, carefully remove the bottom 60 μl and transfer to a new Beckman tube labeled with the sample number. Discard the upper portion of the sample (impure VLDL). Between samples rinse the Hamilton syringe with distilled water.
?. Add 60 μl KBr solution (density = 1.12 g/ml) to make a final density of 1.063 g/ml) and mix 5 to 6 times up and down with the same pipette tip.
?. Spin for 18 h overnight in the ultracentrifuge at 70,000 rpm at 4C as above.
?. Using a rinsed 100 μl Hamilton syringe remove the bottom 60 μl to a new Eppendorf tube labeled HDL. Discard the upper portion of the sample containing mostly LDL.
?. Measure cholesterol, triglycerides or phospholipids concentrations in the HDL fraction using their respective protocols.
?. The non-HDL is calculated by subtracting the HDL from the total.The density of the HDL fraction is > 1.063 g/ml
TITLE: Determining the total cell concentration (TCC) in drinking water with flow cytometry
AUTHORS: juerg.sigrist , frederik.hammes 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to stain cells in (drinking) water samples with SYBR Green I for the purpose of total cell concentration measurements and flow cytometric fingerprinting. The protocol is based on the studies of Prest et al. (2013) and Van Nevel et al. (2013). The protocol is applicable for different types of water samples, and based on the flow cytometric equipment, the volumes of samples and reagents can simply be adjusted.</div></div>

[STEPS]
?. Transfer  of the water sample into a labelled 3.5 mL sample tube. Or the corresponding volume when using other sample tubes.
500 µl
?. Add   of SYBR Green I® working solution (1:100 dilution of stock solution) to the tube and vortex briefly. The final SYBR Green I® dilution in the sample is therefore 1:10'000.
5 µl
SYBR Green I® has DNA-binding properties. It is recomended to wear gloves for this step.(see additional infos under "Warnings")
?. Incubate for a minimum of   at  in the dark. Longer incubation is not problematic, but should be limited to a maximum of 30 min.
[Min.]
37 °C
?. Vortex briefly and measure a volume of at least  on a cleaned and calibrated flow cytometer with appropriate settings, using green fluorescence as the main trigger/threshold parameter.
50 µl
?. Distinguish between microbial cells and background noise with the gating feature of the flow cytometry software.
?. Dilute the water sample with 0.1 µm filtered water (filtered bottled water, original sample or buffer) if a concentration of more than 1 x 106 cells/mL is expected. Multiple samples can be prepared and processed simultaneously.
?. Create a density plot of green fluorescence (520 nm) vs. red fluorescence (620+ nm)
TITLE: Immunofluorescence and object-based colocalization analysis
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Immunofluorescence and object-based colocalization analysis

[STEPS]
SECTION: Immunofluorescence
1. 



For immunostaining, DIV17-21 neurons were fixed in 100% ice-cold methanol solution
for 15 min at room temperature, followed by extraction in 0.1 % Triton X-100
for 10 min, blocked in 10% BSA for 1 h at room temperature, and incubated
overnight at 4°C
with primary antibodies. We found that the methanol fixation was better than
paraformaldehyde fixation for pSer129 α-syn staining. 





SECTION: Object-based colocalization analysis
4. For α-syn Ser129 and VAMP2 colocalization analysis in cultured neurons, 5-6 regions
of interest (ROIs) of 200x200 pixels were placed on each image. A total of 26
ROIs were used for this analysis. 
SECTION: Object-based colocalization analysis
5. Object-based colocalization analysis was done
using MATLAB (RRID:SCR_001622)( http://www.mathworks.com/products/matlab/). First, automatic puncta detection was done using local maxima. Next, detected puncta are said to have co-localized if the distance between their centers is less than the maximum radius of the two
particles.
SECTION: Immunofluorescence
2. After washing with 1x PBS, neurons were blocked again for 30 min at room temperature and incubated with secondary antibodies (Alexa Fluor antibodies from Invitrogen
(RRID:AB_2633275, RRID:AB_2762824,RRID:AB_2633282) (1:500)
for 1 h at room temperature.
SECTION: Immunofluorescence
3.  Images were acquired at 40X magnification. Z-stack
images were obtained as previously described , and all images were acquired and
processed using the MetaMorph
Microscopy Automation and Image Analysis Software (RRID:SCR_002368)(https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref).