Examples of application of DeadEasy Caspase to address biological questions.

<p>DeadEasy Caspase is used to count the number of apoptotic cells stained with Caspase in wild-type embryos at different stages and in H99 mutants lacking apoptosis. Numbers over box-plots indicate number of embryos analysed per genotype.</p

Parameters that can be altered by the user and the consequences.

<p>This table sows the parameters that the user can modify after accessing through ImageJ theprogrammes written in Java. Any parameter modification will require cycles of validation and further modification by the user, until the desired accuracy for the sample and staining in use is reached.</p

DeadEasy Caspase: the mathematical algorithm.

<p>DeadEasy Caspase detects apoptotic cells in embryos stained with anti-cleaved-Caspase-3, and are characterised by low cytoplasmic signal, high background and volume ≥1.56 µm³. (A) Diagram showing the region of the embryonic VNC (blue) scanned for counting. (B) Caspase histogram. (C) Enlarged images to compare a raw image vs. the result (single confocal 0.25 µm slices shown). (D) Diagram of the algorithm. (E) Images showing the different steps of processing, correlating with each step in the diagram in (D). (F) Examples of bad quality stainings or images that must not be used for cell counting as they will produce false positives.</p

The extent of apoptosis in the Drosophila embryonic VNC.

<p>(A) A Drosophila embryonic VNC. The Region Of Interest (ROI) box in red indicates the areas analysed in (B,C). (B) 3D rendering of confocal stacks of sections through the VNC to show the abundant number of embryonic apoptotic cells stained with anti-cleaved-Caspase-3. (C) Cross section view of the image in (B).</p

Validation of DeadEasy Caspase.

<p>(A) During processing, DeadEasy creates a second stack of confocal images reproducing the identified objects in locations that correspond to those of cells in the original raw stack. By placing the mouse over each of the objects, an identifying number is revealed (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005441#pone-0005441-g002" target="_blank">Figure 2</a>), showing whether a cell is counted appropriately. We compared one cell at a time in this way through multiple stacks. (B) Using a second validation method, a merged stack can be created from the raw images (green) and the processed images with the identified objects (red). Colocalising cells in the merged stack (yellow) indicate the identified cells, green cells are false negatives and red cells are false positives.</p

How DeadEasy software works.

<p>How DeadEasy software works.</p

Properties of cells counted by DeadEasy Caspase.

<p>Properties of cells counted by DeadEasy Caspase.</p

Loss and gain of DNT function induce changes in bouton morphology and number and axonal terminal length at the NMJ.

<p>(A,C, F) Third instar larval NMJ preparations, showing synaptic boutons by the colocalisation of pre-synaptic HRP (magenta, also labelling the axons) and post-synaptic Dlg (green). (A’, C’) High magnification details of (A,C) showing an increase in bouton number (A’, <i>spz</i>²), and stretches of Dlg signal along the axons with no boutons (A’ <i>spzCK</i>, C’) (B,D) Quantification of bouton number normalized to muscle surface area (MSA). (E-G) Axonal terminal length increases upon alterations in DNT function, (F) shows the muscle 6,7 NMJ; quantification in (E,G) shows axonal terminal length normalized over MSA. Numbers within bars indicate n=number of NMJs analysed. Significance: (B,D) One Way ANOVA: p=0.000 and p=0.001, and post-hoc Dunnett corrections. (E) One Way ANOVA: p=0.029. (G) One Way ANOVA: p<0.000. Asterisks indicate comparisons to control yw, Dunnett post-hoc tests, * p<0.05, **p<0.01, ***p<0.001. GAL4 drivers: <i>elavGAL4</i> (neurons) or <i>24BGAL4</i> (muscle). Scale bars: 20 µm. For raw data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone-0075902-t001" target="_blank">Table 1</a>, for detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p

Normal synaptic transmission, but reduced spontaneous release in the mutants.

<p>(A,E) Five representative traces showing evoked excitatory junction potentials (EJPs) evoked by 1 Hz stimulation and low extracellular calcium, recorded from muscle 4, 6 or 7 of wild-type (yw), <i>spz</i>² and <i>DNT1</i>^<i>55</i><i>DNT2</i>^{<i>e03444</i>} mutant third instar larvae. Loss of <i>spz, DNT1</i> and <i>DNT2</i> has no effect on either the time course (A,E) or the amplitude (D,H) of the response. (B,F) Four representative traces of recordings of spontaneous neurotransmitter release (mEJPs). (C,G) The frequency and amplitude of the spontaneous mEJPs recorded from muscle 4 are both reduced in <i>spz</i>² mutants; quantal content is not affected (D). Spontaneous mEJPs are not affected in muscle 6,7 (G). Student-t tests for pair-wise comparisons, *p<0.05, **p<0.01, ***p<0.001. For detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p

Loss of DNT function causes a reduction in active zones.

<p>(A,B) Third instar larval NMJ preparations showing active zones labelled with nc82/Brp (green) over HRP+ axons (magenta). (B) There is a reduction in active zones per bouton in the mutants (higher magnification details from A). (C, E) Number of active zones per bouton: (C) counted manually and (E) using DeadEsy Synapse (as total nc82/Brp voxel occupancy per bouton), one bouton was chosen randomly from each NMJ. (D, F) Number of active zones per NMJ terminal: (D) counted manually and (F) active zone density per NMJ terminal, measured using DeadEasy Synapse (as total nc82/Brp voxel occupancy per terminal), decreases in the mutants. (G) Rescue: the reduction in active zone number per bouton and DeadEasy voxel volume per bouton at the muscle 6,7 NMJ in <i>DNT1</i>^<i>41</i><i>DNT2</i>^{<i>e03444</i>} double mutants is rescued with the over-expression of the <i>DNTs</i> in neurons. (H) Rescue: the number of active zones per NMJ terminal and of total active zone occupancy in the 6,7 NMJ in <i>DNT1</i>^<i>41</i><i>DNT2</i>^{<i>e03444</i>} double mutants is rescued with the over-expression of the <i>DNTs</i> in neurons. Numbers within bars indicate n=number of NMJs analysed. Asterisks indicate comparisons to <i>yw</i> control, all samples were analysed using One Way ANOVA and post-hoc Dunnett tests: *p<0.05, **p<0.01, ***p<0.001. Scale bars: 20 µm. For raw data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone-0075902-t001" target="_blank">Table 1</a>, for detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p