eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved <em>Drosophila melanogaster</em> transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.</p>

[BEFORE_START]
<p><strong>Reagents and Supplies</strong></p>
<p><strong> </strong></p>
<ul>
<li>Cy2 Goat α-Mouse Jackson Immuno Research. # 115-225-166</li>
<li>Cy3 Goat α-Rabbit Jackson Immuno Research. # 111-165-144</li>
<li>ATTO 647N Goat α-Rat IgG (H&amp;L) Antibody. Rockland. # 612-156-120</li>
<li>DPX Mountant for Microscopy. Electron Microscopy Sciences. # 13512, 500 mL</li>
<li>Ethanol, ACS reagent, &gt;99.5% (200 proof). Sigma Aldrich. # 459844-1L</li>
<li>GS – Goat Serum. Life Technologies. # 16210-064, 100 mL</li>
<li>Kodak Photo-Flo 200 Solution. Electron Microscopy Sciences. # 74257</li>
<li>nc82 – Mouse α- Developmental Studies Hybridoma Bank.  # nc82-s</li>
<li>PBS - Phosphate Buffered Saline, 1X<strong>. </strong> # 21-040</li>
<li>PFA – Paraformaldehyde. 20% PFA. Electron Microscopy Sciences. # 15713-S</li>
<li>Poly-L-Lysine. Sigma Aldrich. # P1524-25MG</li>
<li>Protein LoBind Microcentrifuge Tubes. Eppendorf. # 022431102</li>
<li>Rat α-FLAG Tag (DYKDDDDK Epitope Tag). Novus Biologicals. # NBP1-06712</li>
<li>Rabbit α-HA Tag. Cell Signal Technologies. # 3724S</li>
<li>S2 – Schneider’s Insect Medium. Sigma Aldrich. # S01416</li>
<li>Triton X-100. Sigma Aldrich. # X100</li>
<li>Xylenes. Fisher Scientific. # X5-500</li>
</ul>

[GUIDELINES]
<ul>
<li>All tissues and solutions are at room temperature (RT), unless noted. Always protect tissue from light exposure.</li>
<li>For details on dissection and fixation see FlyLight Protocol - Adult Dissection and 2% Fixation.</li>
<li>For mounting and embedding instructions refer to FlyLight Protocol – DPX Mounting.</li>
<li>Protocols at <a href="https://www.janelia.org/project-team/flylight/protocols" target="_blank" rel="noopener noreferrer">https://www.janelia.org/project-team/flylight/protocols</a></li>
</ul>
<ul>
<li>Reporter Genotype: w; brp-SNAP / CyO; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1</li>
<li>For details on polarity constructs, please refer to Aso et al. 2014. <a href="http://elifesciences.org/content/3/e04577" target="_blank" rel="noopener noreferrer">http://elifesciences.org/content/3/e04577</a></li>
</ul>

[STEPS]
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TITLE: Lentivirus production in HEK293FT with calcium-phosphate method 
AUTHORS: Ulduz S. Afshar

[DESCRIPTION]
To produce lentivirus, HEK293FT cells are transfected with lentiviral vector and packaging plasmids using thecalcium-phosphate method.

[STEPS]
SECTION: Day1
1. Seed 6 million HEK293FT in a 10cm plate with 10 ml complete medium (DMEM+10%FBS+1% pen-strep+1% MEM-NEAA)
SECTION: Day 2
2. Add 25 μl from 10 mM Chloroquine (Final Concentration:25μM) to 9 ml of complete medium. (To avoid lysosomal degradation of the transfected DNA)
SECTION: Day 2
3. While preparing the plasmid mix, keep the plates inside the incubator.
SECTION: Day 2
4. In 2ml tubes, prepare the transfection mixture in the following order:
 
 

SECTION: Day 2
5. 500 μl of ice-cold HBS-HEPES buffer saline is added to the mix in a slow dropwise manner.
SECTION: Day 2
6. The Solution was further mixed by passing the tube on a tube rack for 30-45 seconds. until the mixture is full of bubbles.
SECTION: Day 2
7. After 5-7 minutes at RT the mixture was added to the plate drop by drop and with a
circular motion to cover the surface of the plates. 



SECTION: Day 2
8. After 6 hours, the medium was changed.
SECTION: Day 4
9. The medium was collected 48 hours post-transfection and filtered through 0.45 μm filter
SECTION: Day 4
10. The lentivirus-containing medium was aliquoted and stored at -80°C for later use.
TITLE: U Mass - Cholesterol (Total)
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of total cholesterol reflect cholesterol metabolism and are associated with cardiovascular disease.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Cholesterol (Total) test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Nest Building Test
AUTHORS: daniel.dautan, Per Svenningsson, Valina L. Dawson, Ted Dawson, Hanseok Ko

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

[STEPS]
2. Place a paper tissue folded into a 5x5 cm square. 
1. Singly house mice in individual, clean cages.
3. Following 48h the bedding was score between 0 to 5 as follows:
    0- The mice did not touch the paper square.
    1- The mice unfolded the paper square but did not tear it up.
    2- The mice unfolded and started to tear down <10% of the paper square.
    3- The mice tear down the paper square <50%.
    4- The mice tear down the paper square, but the bedding is not perfect.
    5- Perfect bedding.
TITLE: alternative PCR protocol for gene COXI neo-caledonian freshwater micro-crustaceans 
AUTHORS: Coline Royaux, Nicolas Rabet, Céline Bonillo, Frédéric Busson

[DESCRIPTION]
This PCR protocol has been used to amplify the Cytochrome Oxydase I gene of neo-caledonian genera Boeckella, Lynceus, Latonopsis, Eulimnadia and Streptocephalus with several primers.

[STEPS]
1. Prepare the mix for the PCR depending on the quantity of DNA samples you want to amplify
 

 



1.1. For each well, mix 13.22 µL, 2 µL, 0.5 µL and 0.28 µL. This mix is hereafter named "intermediary mix".
 


1.2. Add your primers to the mix, 0.5 µL 10 picomolar (pM) and 0.5 µL 10 picomolar (pM)  for each well. This mix is hereafter named "final reagent mix".
 

 



2. In each well, deposit 18 µL and 2 µL unless for the negative control.
For the positive control, use the DNA of the species on which the primer has been designed.

3. Close the PCR plate with a heated aluminium foil and centrifuge it enough to make sure the reagents are all in the bottom of the well.
4. To amplify targeted DNA sequences, place your plate in the thermocycler, set and launch the cycle as described in the following sub-steps
4.1. 5 min at 94 °C 

4.2. 40 cycles of the following steps :
30 s - 1 min at 94 °C 
30 s - 1 min at 45 °C - 52 °C 
1 min at 72 °C 

4.3. 8 min at 72 °C 

4.4. Infinite timing at 20 °C so the samples are well preserved and you have some time to pick it up

5. To know if your amplifications worked properly, you now have to proceed to a gel electrophoresis of your PCR products
5.1. To prepare the gel,
 

 mix 0.5 mg and 30 mL, heat the preparation in the microwave for a few moments until it is homogeneous (you can agitate the preparation). Wait until the preparation is cold enough to touch its container and add 0.5 µL.
5.2. Pour the preparation in a mold and let the gel polymerize with a bar to form wells to deposit your PCR products
5.3. Deposit the gel in the electrophoresis tank
5.4. Fill each well with 3 µL and  0.5 µL - 1.5 µL , don't forget to save a well to deposit 1.2 µL 

5.5. Start electrophoresis in TAE buffer for 15 min at 200 Volts

5.6. Look at your gel under Ultra Violet light to see the results
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Spatial heterogeneity of soil respiration and its temperature sensitivity poses great challenge to accurately estimating carbon flux in global carbon cycling, which have been researched mostly on flatlands but few times in hillslope ecosystems. On an eroded slope (35°) of the semiarid Loess Plateau, soil respiration, soil moisture and soil temperature were measured<em> in situ</em> at upper and lower slope positions in triplicates from 2014 untill 2016, with soil biochemical and microbial properties also being determined.</p>

[STEPS]
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TITLE: Membrane Fractionation
AUTHORS: Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Membrane Fractionation</span><span> (Ultracentrifugation)</span></div></div>

[STEPS]
?. seed cells for  in a 10 cm dish at a density of 6 x 106 cells per plate
?. wash cells with 1X PBS for 2 times
?. scrape cells of the dish and harvest in 1 ml pf PBS + PI buffer
?. Centrifuge: 1000 34, 15 min
?. resuspend cells in 500 µl PBS + PI buffer
?. sonicate 2 times at 30Hz for 15 ON-OFF intervals of 10 s each (1 s pulse on/off).
?. collect supernatant as cytosolic fraction
Centrifuge: 340000 34, 15 min, 4 10
?. was pellet 3 times with PBS + PI buffer
?. Centrifuge: 340000 34, 15 min, 4 10
?. disolve pellet in  1x PBS +PI+ 1% Trtiton
500 µl
?. collect supernatant as membranous fraction
Centrifuge: 340000 33, 15 min, 4 10
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Trace metals solution to be used in ESAW Media for Marine Phytoplankton </p>

[BEFORE_START]
<p>Trace Metal Solution amounts are per ONE LITER, MilliQ H<sub>2</sub>O. Prepare stock volumes according to individual need.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Sueoka's High Salt Medium is used for photoautotrophic growth of the green alga <em>Chlamydomonas reinhardtii</em>. The medium is also known as HS or HSM.</p>
<p> </p>
<p>Source:</p>
<p>Sueoka, N. (1960) <em>Proc. Natl. Acad. Sci. USA</em> <strong>46</strong>, 83-91.</p>
<p>http://www.chlamycollection.org/Sueoka.html</p>

[STEPS]
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TITLE: Calf-Intestinal Alkaline Phosphatase Treatment in situ-Killinger 2024
AUTHORS: Bryan Killinger, Solji Choi

[DESCRIPTION]
Alpha-synuclein phosphorylated at serine 129 (PSER129) occurs in two pools, non-aggregated (physiological) and aggregated (disease). This protocol allows for the selective dephosphorylation of non-aggregated PSER129 and enhances the specificity and sensitivity immunodetection of aggregated PSER129. Thus, this protocol can be used to differentiate physiological from aggregated PSER129.

[STEPS]
SECTION: Day 1
1. Wash free-floating tissue (3 x 10 minutes) in dilution media.
 
Dilution media:

 
 
SECTION: Day 1
2. Incubate the samples with 1% Triton X-100 in DM for 10 min.

SECTION: Day 1
3. Wash in DM 10 min.

SECTION: Day 1
4. Wash the tissues in CIAP buffer (2x10 minutes).

CIAP buffer: 
 
 
SECTION: Day 1
5. Incubate the tissues with CIAP at a dilution of 1:333 for 1440 min at 37 °Con a shaker. 

CIAP concentration per bottle: 20 u/μl (Promega, Cat.# M2825).

In 500 µL CIAP buffer, add 1.5 µL CIAP (30 units). 

SECTION: Day 2
6. Wash in DM (2 x 10 minutes).
SECTION: Day 2
7. Heat water bath to 80 °C-85 °C  90 min before the antigen retrieval step. 

SECTION: Day 2
8. Place the dish containing sodium citrate buffer in the water bath and heat it for 10 min.

Sodium Citrate Buffer, pH 6.0 (1L): 2.94 g Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water. pH 6.0. Add 0.5 mL Tween-20. Mix well.

SECTION: Day 2
9. Wash the tissues in sodium citrate buffer 10 min.

SECTION: Day 2
10. Incubate the tissues in the heated sodium citrate buffer for 30 min.

SECTION: Day 2
11. Cool the dish containing tissues to Room temperature (at least 20 min). 

SECTION: Day 2
12. Wash in DM for 10 min x 2 times.
SECTION: Day 2
13. Tissues are now ready for downstream assays.
SECTION: Day 1
1.1. Wash free-floating tissue 10 min in dilution media (1/3).

SECTION: Day 1
1.2. Wash free-floating tissue 10 min in dilution media (2/3).

SECTION: Day 1
1.3. Wash free-floating tissue 10 minin dilution media (3/3).

SECTION: Day 1
4.1. Wash the tissues in CIAP buffer 10 min  (1/2).

SECTION: Day 1
4.2. Wash the tissues in CIAP buffer 10 min  (2/2).
SECTION: Day 2
6.1. Wash in DM 10 min (1/2).

SECTION: Day 2
6.2. Wash in DM 10 min (2/2).
SECTION: Day 2
12.1. Wash in DM for 10 min  (1/2).

SECTION: Day 2
12.2. Wash in DM for 10 min(2/2) .
TITLE: BGISEQ-500 Sequencing
AUTHORS: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> BGISEQ-500 is a new desktop sequencer developed by BGI. Using DNA nanoball and combinational probe anchor synthesis developed from Complete Genomics™ sequencing technologies, it generates short reads at a large scale. </div></div>

[STEPS]
?. Test the quality of the sequencing library (see protocol for library preparation) by the Qubit® ssDNA Assay Kit and homogenized at 6ng total amounts.
?. Carry out Rolling circle amplification（RCA）for 10 minutes in an 80 ul reaction volume with pure water, buffer and DNB polymerase
?. Add 20 ul DNBs stopping buffer to stop the RCA reaction
?. Check the quality of the DNBs using the Qubit® ssDNA Assay Kit , concentration should be above 10 ng/µL
?. Add 33ul DNBs loading buffer to the DNBs product from the last step, and place it in the BGIDL-50 (the DNBs loading machine)
?. install the sequencing chip and selected the DNBs loading process (Version: sample load 2.0) to load the DNBs.
?. after loading, take the sequencing chip out and install in the BGISEQ-500 （DNBSEQ-G50）sequencing machine
?. load the reagent sequencing kit and open the sequence control software (Version 1.1.0.10003), and select the sequence process Version 1.0.06 and Zebracall process Version 0.5.0.13875 for sequencing.
?. Sequencing is initiated after the sequencing reagents preloaded and sequencing chip was installed, and this process is finished in about 72 hours. When the whole sequencing was finished, the binary file with bases and quality score were converted into FASTQ format with Phred+33 quality score.
TITLE: SMARTerV4 (1x) Amplification for single-cell or  single-nuclei RNASeq Protocol
AUTHORS: Allen Institute

[DESCRIPTION]
Protocol to generate full-length cDNA from single cells, or nuclei, using Takara SMARTer V4.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Generation of knockout and rescue cell lines using CRISPR-Cas9 genome editing
AUTHORS: William  Hancock-Cerutti, Jun  Hyun Park, Pietro  De Camilli

[DESCRIPTION]
This protocol describes the genetic modification of cultured cells using CRISPR-Cas9, including synthesis of reagents, transfection, selection and screening of single clones, and sequencing of genomic DNA to confirm mutations. In addition, this protocol describes CRISPR-Cas9 mediated repair of single base deletions or insertions by CRISPR-Cas9.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a modified protocol for Qiagen's DNEasy Blood and Tissue kit for non-destructive DNA extraction of minute insects and other organisms. It was optimized for parasitic wasps (0.5-2mm size).</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The development of self-microemulsifying drug delivery systems (SMEDDS) was obtained from pseudoternary phase diagram construction. This protocol demonstrates how to construct pseudoternary phase diagram. Tween 20, Tween 80, or Triton X-100 was used as a surfactant and absolute ethanol, propylene glycol, or isopropanol was used as a cosurfactant.</p>

[STEPS]
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TITLE: Complexing Sodium Oleate for use in insulin secretion
AUTHORS: Aliya Spigelman, Mourad Ferdaoussi, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for complexing of sodium oleate to be used in insulin secretion experiments. </div></div>

[STEPS]
?. [Preparation of 10% w/v BSA]
Weigh   of fatty acid free BSA (Sigma A6003 )
1 g
?. [Preparation of 10% w/v BSA]
Add BSA into a beaker  with  of ultrapure water
10 mL
?. [Preparation of 10% w/v BSA]
Stir BSA and water until all the BSA has dissolved. *Warming at   may help dissolve BSA.
37 °C
?. [Preparation of 10% w/v BSA]
Filter BSA solution with 0.22μM filter. This can be made in advance and stored at .
4 °C
?. [Preparing 150mM stock of sodium oleate]
Weight approximately   of sodium oleate (MW 305g/L). *It should be noted that sodium oleate is very hydrostatic. Weigh approximately the amount indicated above, then calculate the volume of solvent needed to create a 150mM solution. 50% of this volume is then added in step 6, and 50% in step 8. *This must be made fresh the day of your experiment.
45.7 mg
?. [Preparing 150mM stock of sodium oleate]
Add(or as calculated) of 100% Ethanol and vortex
500 µl
?. [Preparing 150mM stock of sodium oleate]
Using a heat block, heat at   for
65 °C
?. [Preparing 150mM stock of sodium oleate]
After 15 minutes the sodium oleate should be solubilized. Add  (or as calculated) of ultrapure water. No remaining undissolved solute should be visible at this time.
500 µl
?. [Preparing 150mM stock of sodium oleate]
Vortex and continue to heat solution at  for .
65 °C
?. [Preparing 150mM stock of sodium oleate]
Vortex and hold the sodium oleate at  until complexed with BSA.
65 °C
?. [Complexing oleate with BSA (5:1)]
*Below numbers are based on  of solution. Adjust volume as necessary for each experiment*Warm 2 tubes of  of 10% BSA for  in a  water bath. One tube will be for your control group. One group for your oleate group.
10 mL
670 µl
37 °C
?. [Complexing oleate with BSA (5:1)]
For control group, add  of 50% ethanol to one pre-warmed BSA tube from step 11.
33 µl
?. [Complexing oleate with BSA (5:1)]
For oleate group add  of  oleate stock to one pre-warmed BSA tube from step 11.*Important note* It is very important to keep BSA warm when adding oleate. If hot plate and water bath are not beside each other, place tube with BSA in a beaker containing 37°C of water.
33 µl
?. [Complexing oleate with BSA (5:1)]
Incubate both control and oleate tubes in at shaking water bath () for .*Check oleate+BSA tube after approximately  to make sure the solution isn't cloudy. If it is cloudy, discard and restart at step 11.*If you are using KRBH for your secretion experiment, make sure it is prepared prior to the end of this hour.
37 °C
?. [Complexing oleate with BSA (5:1)]
After 1 hour add  of warm KRBH (or solution to be used for experiment) to each tube. Add glucose or other compounds as needed. Solution must be kept at  and used on the same day it is made.
9.3 mL
37 °C
TITLE: Pichia pastoris strain and growth condition
AUTHORS: iGEM Dusseldorf

[STEPS]
?. [Preparation ]
Grow Pichia pastoris cells on agar plate containing desired antibiotic ( non-shaking 30°C 72 hours )
?. [Day one]
Cultivation
?. [Day one]
Add growth medium (BMGY) to baffled flasks
3ml-10ml growth medium in 30mL-100mL flasks to ensure sufficient aeration
?. [Day one]
Inoculate growth medium with single positive colony
?. [Day one]
Incubate at 30°C at 225rpm overnight
30 °C
?. [Day two]
Gene expression
?. [Day two]
Transfer cultures to sterile 50mL falcon tubes
?. [Day two]
Centrifuge and decant supernatant
Centrifuge: 2000 34
?. [Day two]
Add 10mL of sterile BMMY to used baffled flasks. Swirl and transfer to corresponding falcon tube
?. [Day two]
Vortex falcon tube
?. [Day two]
Centrifuge falcon tube at
Centrifuge: 2000 34
?. [Day two]
Decant supernatant and add 10 mL of fresh BMMY
?. [Day two]
Vortext solution and transfer to original flasks
?. [Day two]
Incubate at 225 rpm overnight
30 °C
?. [Day three]
Continued expression
?. [Day three]
Add 50 µL sterile 100% methanol to flask
?. [Day three]
Incubate at 30°C at 225 rpm overnight
30 °C
?. [Day four]
Cell harvesting
?. [Day four]
Transfer cells to 50 mL falcon tubes from second day and harvest by centrifuging at
Centrifuge: 2000 34
?. [Day four]
Decant supernatant and resuspend cell wall containing pellet in 1 mL water.
?. [Day four]
Transfer to pre-weighed 2 mL tube
?. [Day four]
Centrifuge, decant water and use cells for desired experiments
Centrifuge: 10000 34
?. [Alternatives ]
For each step where media was added, only 3 mL instead of 10 mL were added.All centrifugation steps were performed at 3.000 x g instead of 2.000 x g and only 20 μL of sterile 100 % methanol were added for methanol induction
?. [Alternatives ]
Grow liquid cultures with starting volume of 3 mL in similar procedure
From now on: use plastic tubes
?. [Alternatives ]
Store harvested cells at -20°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
On a high speed slaughter line for pigs, skin scratches were separately scored in the posterior region (defined as the area including the hind legs and the tail) and the anterior one (as the remaining area), while the whole carcass was examined for external hematomas. Chronic ear and tail lesions referable to the rearing phase, and bursitis were recorded as retrospective welfare indicators.

[BEFORE_START]
Get the material to easily record the data (prepared paper sheet or tablet).

[GUIDELINES]
To score acute traumatic lesions (scratches) on slaughtered pigs, the carcass was divided into two parts: the “posterior” region, which included the hind legs and the tail, and the “anterior” region defined as the remaining area (starting from the loin up to the front limbs, the head and the ears). In order to easily scan the carcasses during their rapid passage on the dressing line (480 pigs/h), a 3 point scoring system for each of the two carcass regions was used: score 0, up to one scratch or bite; score 1, from two to five scratches or bites; score 2, more than five scratches or bites or any wound which penetrates the muscle. In addition, external hematomas were recorded as presence or absence (binary score).
The presence of lesions with a chronic onset such as notches, necrosis, bites and scars were evaluated on ears and tail as welfare indicators referable to the rearing cycle, as well as the presence of bursitis. Bursitis was only evaluated on the forelimbs, because hind legs were not completely visible to the observer since the carcass was hung upside down above the inspection area. These lesions were recorded as present or absent (binary score) regardless of the size, in accordance with the possibility to be detected in the few seconds allocated to the observer. For ear and tail lesions, only the older ones were recorded as retrospective welfare indicators, since the recent ones were already recorded with the scratches scoring system as pre-slaughter fighting or transport lesions, so that recent hyperaemic or fresh bleeding lesions were not recorded as retrospective indicators. Post-mortem artefacts due to the slaughtering process were excluded on the basis of the absence of pathological or physiological changes occurred in-vivo such as, for example, necrosis or scarring.
In order to get a representative batch sample, about 100 carcasses per batch were recorded.

[STEPS]